Protein clean-up technical handbook Dialysis • Desalting • Detergent removal • Concentration • Endotoxin removal Contents Protein clean-up technical handbook

Contents

Introduction 2 Protein concentration using ultrafiltration membranes Overview 30 Protein dialysis using regenerated cellulose membranes Selecting the right membrane 31 Overview 4 Membrane performance data 32 Membrane performance data 6 Selecting the right format 34 Selecting the right format for sample processing 8 Pierce Protein Concentrators, 0.5 mL 35 Pierce 96-well Microdialysis Plate 9 Pierce Protein Concentrators, 6 mL 35 Slide-A-Lyzer MINI Dialysis Devices 10 Pierce Protein Concentrators, 20 mL 36 Original Thermo Scientific Slide-A-Lyzer Dialysis Cassette 11 Pierce Protein Concentrators, 100 mL 36 Slide-A-Lyzer G2 Dialysis Cassettes 12 Slide-A-Lyzer Dialysis Flasks 13 Endotoxin removal using affinity chromatography SnakeSkin Dialysis Tubing 14 Overview 37 Resin performance data 38 Protein desalting using gel filtration resins Selecting the right format 40 Overview 15 Spin columns 40 Resin performance data 17 Loose resin 41 Selecting the right format for sample processing 21 Endotoxin quantitation kit 41 Zeba Spin Desalting Columns 22 Zeba Spin Desalting Plates 23 Ordering information 42 Zeba Desalting Chromatography Cartridges 24 References 47 Detergent removal using chromatography resins Overview 25 Protein research handbooks 48 Resin performance data 26 Selecting the right format for sample processing 28 Spin columns, loose resin and kits 29 96-well spin plates 29

2 Introduction Protein clean-up technical handbook

the membrane barrier in a centrifuge tube, concentrating Endotoxin removal Overview of protein clean-up methods the macromolecules (e.g., proteins) in the remaining Endotoxin contamination is a common problem with solution (retentate). For buffer exchange (diafiltration), the recombinant proteins purified from gram-negative bacteria concentrated solution is diluted and concentrated multiple such as E. coli. Traditional endotoxin removal methods times until the desired state is achieved. The easy-to-use include anion-exchange chromatography, ultrafiltration, Thermo Scientific™ Pierce™ Protein Concentrators contain membrane-based chromatography, and Polymixin B affinity a high performance PES (polyethersulfone) membrane ligand. These methods are limited by specificity, capacity Flat dialysis tubing composed of cellulose acetate or that enables fast processing and excellent protein recovery or reusability. Thermo Scientific™ Pierce™ High Capacity The first step in protein analysis is regenerated cellulose was introduced in the 1950s. This for samples. Endotoxin Removal Resin selectively binds and removes format requires preparation and is cumbersome and endotoxins from protein, peptide and antibody samples cellular extraction. Following lysis, difficult to handle. Thermo Scientific™ dialysis products Thermo Fisher Scientific offers a variety of specialty devices using a modifiedε -poly-L-lysine [poly(ε-lysine)] affinity ligand. are essentially ready to use and are designed to eliminate and resins that simply and efficiently desalt, exchange buffer, and depending on the next step potential sample leakage and maximize ease of use for and remove detergents from samples. In addition, if the specific applications. protein sample is too dilute for further processing or analysis, in the workflow, the protein extract the sample can be concentrated quickly using centrifugal Desalting concentrators (Table 1). may require further clean-up or Size exclusion chromatography (also known as gel filtration or molecular sieve chromatography) can be effectively Table 1. Protein clean-up selection guide enrichment during downstream utilized for protein desalting (removal of salt from a sample). A resin is selected with pores that are large enough to trap Dialysis devices, Desalting spin columns, Detergent removal spin processing, using techniques cassettes, 96-well 96-well spin plates columns and 96-well small contaminants (e.g., salts), but too small for the protein plates and tubing and cartridges spin plates Concentrators such as dialysis, desalting, of interest to enter. The larger, faster proteins separate from the slower, smaller molecules and can be collected first. concentration, or contaminant- The Thermo Scientific™ Zeba™ desalting products contain a unique resin that enables exceptional desalting and protein specific removal. recovery, and are available in convenient spin columns and plate formats that allow samples to be processed in minutes. Historically, mechanical disruption has been used to lyse Sample volume 10 μL–250 mL 2 μL–4 mL 25 μL–1 mL 100 μL–100 mL cells and tissues; however, detergent-based solutions have Detergent removal processing range more recently been developed to efficiently lyse cells and Detergent removal has traditionally utilized a variety of Sample processing time 2–24 hrs 5–10 min 15–20 min 5–30 min* enable the separation of subcellular structures without methods including dialysis, ion exchange chromatography, (volume dependent) requiring physical disruption. Many detergents, salts sucrose gradients, or acid or acetone precipitation. However, Key applications Buffer exchange, desalting, Desalting, buffer exchange, Removal of anionic, Sample concentration, and other molecules used in or generated during protein all these methods can be labor- and/or time-intensive, virus purification removal of unincorporated nonionic and zwitterionic desalting, buffer exchange extraction or purification may have adverse effects on or detergent-specific. The proprietary Thermo Scientific™ label detergents protein function or stability, or interfere with downstream Detergent Removal resins enable the efficient, rapid and Recommended Purified protein Lysate or purified protein Purified protein or peptide Lysate or purified protein applications; therefore it may be necessary to remove effective extraction of a wide variety of detergents (ionic, sample type mixture or reduce these contaminants using one or more of the nonionic and zwitterionic) that are commonly used in protein Compatible with Yes No No No following methods. extraction, purification and biological assays. viscous samples?

Sample diluted during Possible No No No Dialysis Concentration processing?

Dialysis is a separation method that utilizes selective Protein concentration utilizes a semi-permeable membrane Gamma-irradiated Yes No No No diffusion through a semi-permeable membrane to remove to separate macromolecules from low molecular weight options available? small contaminants or exchange buffers from proteins in compounds. Unlike dialysis, which relies on passive * For 10K MWCO (device dependent—times may vary for other MWCOs) solution. Proteins that are larger than the membrane pore diffusion, concentration is achieved by forcing solutions size are retained on one side of the membrane while small through the membrane by centrifugation. Solvents and Learn more at thermofisher.com/proteincleanup molecular weight contaminants diffuse freely through the small molecular weight molecules pass through the membrane and approach an equilibrium concentration. membrane pores as the protein solution is forced against

2 3 Protein dialysis using regenerated cellulose membranes Protein clean-up technical handbook

Factors that influence dialysis frequency exchange of the external buffer. The rate of Protein dialysis using regenerated Dialysis is used for separating molecules with significantly dialysis is also directly proportional to the surface area of different molecular weights (typically a 10- to >50-fold size the membrane in relationship to the volume of the sample difference). The diffusion properties of a membrane are and the average distance of the sample from the membrane. cellulose membranes determined by the average or maximum size of its pores, The more that a sample can be spread over a membrane the number of pores and the thickness of the membrane. surface, the faster dialysis will proceed because all Membrane selection is based on the molecular weight molecules in the sample will be closer to the membrane, and Through this process, the concentration of small cutoff (MWCO), which is defined as the average molecular a higher proportion of them will be in direct contact with the Overview contaminants in the sample can be decreased to acceptable weight of a molecule that can no longer diffuse across membrane at any instant. or negligible levels if the external buffer volume is large the membrane, such that it is retained at >90%, as it no Dialysis is a classic solution-based (typically 30–500 times the sample volume). Alternatively, longer fits into the pores. Understanding the significance Optimized products for easier, efficient dialysis desired components in the external buffer solution can be of a membrane’s MWCO and how it behaves enables Using dialysis to separate complex mixtures of separation technique that facilitates slowly brought into the sample. Dialysis is used for a wide selection of the best membrane for a particular dialysis biomacromolecules was established in the 1950s. Many variety of applications including simple salt removal and application. Molecular weight cutoff is not a “defined” value, of the dialysis theories established at that time are the the removal of small, unwanted buffer exchange, removal of labeling reagents, drug binding as diffusion of molecules near but below the MWCO will also cornerstones for contemporary dialysis products. However, studies, cell growth and feeding, and virus purification. be significantly slowed. Molecules with a molecular weight significant improvements have been made to the dialysis compounds from macromolecules that is less than 1/10 of the MWCO rating of the membrane tools of yesterday, including faster preparation time, ease will diffuse most rapidly and reliably across the membrane of use and reliability. Thermo Scientific dialysis products by selective diffusion. In a typical (see data, page 6). A membrane with the proper MWCO are essentially ready to use and are designed to eliminate will prevent loss of proteins of interest and ensure adequate potential sample leakage and maximize ease of use for dialysis application, a sample IgG removal of contaminants. specific applications. The high performance Thermo (160 Å) Scientific™ Slide-A-Lyzer™ cassettes and flasks are designed and a buffer solution are placed Although the membrane and its properties are the primary to maximize surface-area-to-volume ratios (within practical factors that affect dialysis rate, a variety of other factors limits) for different sample volumes. Our broad product on opposite sides of a semi- Pore (24 Å) can also influence dialysis. These include temperature; the portfolio covers a variety of formats and membrane permeable membrane. Sample geometry, concentration, interactions and hydrophobicity molecular weight cutoffs (MWCOs) to accommodate a of the molecules; as well as the volume, agitation and wide range of sample volumes and workflows. 10K molecules that are larger than the Membrane membrane pores are retained on

2-ME the sample side of the membrane, (5 Å) but small molecules diffuse Enlarged view of Slide-A-Lyzer Enlarged view of Slide-A-Lyzer Enlarged view of Slide-A-Lyzer cassette interior cassette interior cassette interior freely through the membrane Figure 1. How dialysis membranes work. A dialysis membrane is a semi-permeable film (usually a sheet of regenerated cellulose) containing various sized pores. Molecules larger than the pores cannot pass through and approach an equilibrium Old dialysate discarded and Old dialysate discarded and the membrane, but small molecules can do so freely. In this manner, replaced with 1,000 mL of replaced with 1,000 mL of dialysis may be used to perform purification or buffer exchange for 100 mM PBS, pH 7.6 100 mM PBS, pH 7.6 concentration with the entire buffer samples containing macromolecules. volume (Figure 1). 2 hours 2 hours

3 mL of 1 mg/mL IgG in 0.1 M Tris IgG would diffuse out if it could, but it is too large to enter IgG inside the cassette remains buffer, pH 7.4 inside a Slide-A-Lyzer the pores in the membrane; therefore, the amount of IgG constant. The Tris buffer inside the Dialysis Cassette (10K MWCO) inside the cassette remains constant. The Tris buffer cassette drops to near undetectable placed in 1,000 mL of 100 mM concentration drops below 0.01 M inside the cassette as levels. The buffer inside the cassette PBS, pH 7.6. the Tris buffer diffuses out and the PBS buffer diffuses in. is 100 mM PBS, pH 7.6.

Figure 2. How the Thermo Scientific Slide-A-Lyzer cassette works.

4 5 Protein dialysis using regenerated cellulose membranes Protein clean-up technical handbook

Membrane performance data Dialysis rates for various formats

Protein recovery by MWCO 100 2K 90 3.5K 2K MWCO 3.5K MWCO 7K MWCO 10K MWCO 20K MWCO 1.25 80 10K 70 20K 100 1.00 60 100 100 100 100 100 Vitamin B12 (1.4K MW) Dialysis device in conical tube 50 Bacitracin (1.4K MW) 75 Conventional dialysis 0.75 T Kinase Peptide (1.7K MW) 40 80 80 80 80 90 Biotin-TPK (1.9K MW) 50 0.50 30 Ce Peptide (2K MW) 20 Insulin A Chain (2.5K MW) 25 0.25 Residual 1M NaCl (%)

Residual NaCl (M) NaCl Residual 10 Insulin B Chain (3.5K MW) 60 60 60 60 80 Insulin (5K MW) % Residual NaCl 0 0 0 Cytochrome C (12.4K MW) 0 1 2 3 4 5 0 1 2 3 4 5 0 5 10 15 20 25 Lysozyme (14K MW) Time (hours) Time (hours) Duration of dialysis (hours) 40 40 40 40 70 Myoglobin (17K MW) % Retention % % Retention % % Retention % Retention % % Retention % A B C

20 20 20 20 60 Figure 4. The rate of removal of NaCl using various dialysis products. of 0.1 mL (0.4 mg/mL cytochrome C containing 1 M NaCl) were dialyzed in NaCl removal from samples was determined by measuring the conductivity the Thermo Scientific™ Pierce™ 96-well Microdialysis Plate against 1.8 mL of the retentate at the indicated times. Panel A: Slide-A-Lyzer MINI Dialysis of water in a 96-well deep-well plate at RT with gentle shaking. The buffer 0 0 0 0 50 Device (10K MWCO, 2 mL) versus conventional dialysis. Bovine serum was changed at 1-, 2- and 3-hour intervals over a 4-hour period. Removal albumin (BSA) samples (2 mL, 0.25 mg/mL in 1 M NaCl) were dialyzed of NaCl was >83% after 2 hours and >99% after 4 hours. Panel C: Proteins Figure 3. Sample retention by the 2K, 3.5K, 7K, 10K and 20K MWCO Thermo Scientific Slide-A-Lyzer Dialysis Cassette membrane. against 45 mL of water in 50 mL disposable conical tubes on an orbital in 200 mL samples containing 1M NaCl were dialyzed at room temperature Individual proteins (1 mg/mL) in either saline or 0.2 M carbonate-bicarbonate buffer, pH 9.4 were dialyzed overnight (17 hours) at 4°C. The ™ ™ ™ ™ shaker (300 rpm) at room temperature. The water was changed once after using Thermo Scientific amount of retentate was estimated using either the Thermo Scientific Pierce BCA Protein Assay or absorption at 360 nm (for vitamin B12). Slide-A-Lyzer Dialysis Flasks in 2K, 3.5K, 10K, 2 hours. Results are the average of two samples. For conventional dialysis, and 20K MWCOs. The dialysis buffer (4 L) was changed after 2 and 5 hours the samples were dialyzed against 2 L of water in a beaker with stirring. (triangles; also at 41 hours for the 2K condition). Greater than 95% of NaCl Greater than 95% of NaCl was removed within 4 hours. Panel B: Samples was removed within 8 to 18 hours (41 hours for the 2K condition). Protein recovery for specific devices

Table 1. High protein recovery is obtained using the 2 mL Table 2. Quantitative sample recovery using a Thermo Scientific™ Thermo Scientific™ Slide-A-Lyzer™ MINI Dialysis Device.† Slide-A-Lyzer™ Dialysis Cassette vs. conventional tubing. Three sample volume batches of water (0.5 mL, 1.7 mL and 3.0 mL) were Membrane chemical compatibility guide Membrane MWCO (K) Protein/Peptide Recovery (%) loaded and recovered per the respective manufacturer’s instructions in a Slide-A-Lyzer Dialysis Cassette or conventional dialysis tubing Note: The following ratings refer to chemical compatibility with the and other chemicals (see asterisks in table) that are listed as being 3.5 Insulin Chain B (3.5 kDa) 90.13 to determine the volumes of recovery. Water volume recovered was regenerated cellulose dialysis membrane. The plastic cassette frame and compatible with the dialysis membrane. Test solvents with a cassette determined gravimetrically. 10 Cytochrome C (12.4 kDa) 94.44 silicone-like gasket may leach, dissolve, deform, or otherwise fail in certain before attempting to dialyze valuable samples. Traditional strong acids and bases, alcohols, aromatic and chlorinated hydrocarbons, 20 Myoglobin (17 kDa) 95 Sample Slide-A-Lyzer dialysis tubing volume Dialysis Cassette % % volume Acetic acid, 25% G Ethyl acetate G* Nitric acid, <5% G † Insulin chain B, cytochrome C and myoglobin (0.25 mg/mL) in either 50 mM sodium phosphate, loaded volume recovery recovery 75 mM NaCl at pH 7.2 or 0.2 M carbonate-bicarbonate buffer at pH 9.4 were dialyzed overnight Acetone G* Ethylene glycol G Nitric acid, >25% N (17 hours) at 4°C. The amount of protein in the retentate was determined using the Thermo 3.0 mL 99.47 92.32 Scientific™ Pierce™ BCA Protein Assay (Cat. No. 23225). Ammonium hydroxide, 1 N F Formaldehyde solution, 30% G Perchloric acid, 25% N 1.7 mL 99.30 93.12 Ammonium hydroxide, 25% F Formic acid, 25% G* Phosphoric acid, 25% F 0.5 mL 98.76 87.51 Ammonium sulfate, 1 M G Formic acid, 100% G* Potassium hydroxide, 1 N N Amyl acetate G* Hexane G* Propylene glycol G Membrane specifications by MWCO Benzene G* Hydrochloric acid, <5% G Sodium hydroxide, 0.1 N G Benzyl alcohol G* Hydrochloric acid, >25% N Sodium hydroxide, 1 N F Specifications Slide-A-Lyzer Membrane MWCO SnakeSkin Tubing Butanol G* Hydrofluoric acid, 25% F Sulfuric acid, <5% G 2K 3.5K 7K 10K 20K 3.5K, 7K and 10K Butyl acetate G* Hydrogen peroxide, 30% G Sulfuric acid, >25% N Membrane composition all membranes composed of regenerated cellulose Carbon tetrachloride G* Iodine solutions N* Tetrahydrofuran G Required hydration 2 minutes 30 seconds 30 seconds 30 seconds 2 minutes none Chloroform G* Isopropyl alcohol G Toluene G* for low-volume samples Dimethylformamide F* Methanol, <50% G* Trichloroacetic acid, <10% F Dioxane F Methyl acetate G* Trichloroacetic acid, >25% N Glycerol content none trace 13% 21% none varies with MWCO Ethanol, 70% G Methyl ethyl ketone G* Trichloroethylene G* Sulfur content 0.169% 0.1–0.15% 0.1–0.15% 0.05% 0.04% 0.1– 0.15% Ethanol, 95% G Methylene chloride G* Xylene G* Heavy metals content trace trace trace trace trace trace Legend: G = Good resistance; F = Fair resistance (pore swelling may occur); N = Not Recommended

* Chemicals known to adversely affect the plastic cassette frame; brief or dilute exposure may be compatible.

6 7 Protein dialysis using regenerated cellulose membranes Protein clean-up technical handbook

Selecting the right format for sample processing Thermo Scientific Pierce 96-well Each device can also be used independently in a 2 mL Microdialysis Plate microcentrifuge tube, or up to 96 samples can be dialyzed Thermo Scientific dialysis products have evolved over enhanced sample protection, and excellent recovery for simultaneously in a standard 96-well deep-well plate using a the last decade to meet a variety of customer needs. Our small to larger volumes. Available in a variety of formats, it’s High-throughput dialysis in a 96-well plate minimal amount of buffer. broad portfolio of high performance tools for dialysis, buffer easy to find the best dialysis product for your application. exchange, and sample clean up enables easy handling, Highlights: • Efficient and rapid dialysis—dialysis completed in Thermo Scientific Slide-A-Lyzer Dialysis Flask, 150–250 mL 2–4 hours with up to 99% salt removal

Thermo Scientific Slide-A-Lyzer • Excellent sample recovery—up to 90% protein Cassettes, 0.1–30 mL recovery after dialysis 250 mL 12–30 mL 70 mL • Ideal for small sample volume dialysis—uses sample volumes from 10–100 μL

30 mL 15 mL 0.5–3 mL • Easy to use—complete sample loading and retrieval with 3–12 mL The Thermo Scientific Pierce 96-well Microdialysis Plate a standard pipette is an automation-compatible system for simultaneously dialyzing between 1 and 96 samples with volumes from • Flexible—detachable 8-unit strips; scalable from 0.5–3 mL 0.1–0.5 mL 0.1–0.5 mL Thermo Scientific Slide-A-Lyzer 10 μL to 100 μL. The dialysis inserts are provided in strips 1 to 96 samples MINI Dialysis Devices, 10–2,000 μL Thermo Scientific SnakeSkin Thermo Scientific Slide-A-Lyzer Dialysis Tubing, 15–100 mL of eight preloaded in a 96-well deep-well plate, but can be G2 Cassettes, 0.1–70 mL • Automation-compatible—plate format conforms to SBS separated easily for use as individual dialysis devices in a Microplate Standard Thermo Scientific Pierce 96-well Microdialysis Plate, 10–100 μL standard microcentrifuge tube. The assembled device is compatible with standard Table 3. Dialysis products selection guide Each microdialysis device has two regenerated cellulose 96-well laboratory equipment and automated liquid-handling membranes separated by <2 mm. This combination of systems, making it an ideal option for high-throughput Pierce 96-well Slide-A-Lyzer Slide-A-Lyzer Slide-A-Lyzer Slide-A-Lyzer Microdialysis MINI Dialysis Dialysis Cassettes G2 Dialysis Dialysis short diffusion distance and large surface area allows for applications. When used according to the method outlined Plates Devices (original) Cassettes Flasks rapid dialysis. In addition, the small distance between the in Figure 5, the Pierce 96-well Microdialysis Plate enables membranes allows highly efficient sample recovery using the removal of low molecular weight contaminants, buffer standard laboratory pipettes. The dialysis chambers come exchange and desalting within two to four hours (Figure 4, in strips of eight units that can be easily separated allowing Panel B.) with typical protein recoveries of >90%. flexibility in the number of units needed per experiment.

External float required? No No Yes No Optional

Method of sample Pipette Pipette Syringe Pipette or syringe Pipette addition/removal

Volume range 10–100 μL 10 μL–2 mL 0.1–30 mL 0.25–70 mL 150–250 mL OR

# of devices with 1 3 4 5 1 different volume 1. Remove one or more devices, as needed. 2. Add dialysis buffer to a deep-well plate 3. Insert an upright pipette tip filled with sample capacities If only one device is required, break it (≤1,800 µL) or a 2 mL microcentrifuge into the round opening (see arrow). Slowly carefully from the 8-segmented cartridge. tube (≤1,400 µL) and set aside. add the sample (10–100 µL). Molecular weight 3.5K, 10K 2K (0.1 mL), 3.5K, 2K, 3.5K, 7K (0.1, 2K, 3.5K, 7K (0.1, 2K, 3.5K, 10K, 20K cutoffs (MWCOs) 10K, 20K 0.5 mL), 10K, 20K 0.5 mL), 10K, 20K

Automation compatible? Yes No No No No OR Integrated buffer Yes Yes, except No No No reservoir? 0.1 mL size

Gamma-irradiated No No Yes, 10K only Yes, 10K only No 4. Place device into the deep-well plate or 5. Dialyze to remove low molecular weight 6. Remove device from plate or tube and recover 2 mL microcentrifuge tube containing buffer. compounds (1 hour to overnight). sample by inserting upright pipette tip into options? round opening of device and slowly withdrawing the sample. Molecular weight cutoff No Yes Yes Yes Yes (MWCO) color coded? Figure 5. Protocol summary for the Thermo Scientific Pierce 96-well Microdialysis Plate.

8 9 Protein dialysis using regenerated cellulose membranes Protein clean-up technical handbook

Thermo Scientific Slide-A-Lyzer contaminant removal, buffer exchange and desalting can be Original Thermo Scientific The Slide-A-Lyzer Dialysis Cassettes are available in five MINI Dialysis Devices accomplished within 4 to 8 hours with high protein recovery. Slide-A-Lyzer Dialysis Cassettes membrane MWCOs (2K, 3.5K, 7K, 10K, and 20K) and in The recommended sample volume ranges for each device four different sizes for dialyzing sample volumes between Self-contained devices for sample volumes are 10 µL–100 µL (0.1 mL) , 50–500 µL (0.5 mL) and Secure and convenient alternative to 0.1 and 30 mL. Slide-A-Lyzer Dialysis Cassettes can as small as 10 μL 200–2,000 µL (2 mL). dialysis tubing be used for a wide range of applications, including low molecular weight contaminant removal, buffer exchange, Highlights: desalting, and sample concentration. • Excellent sample recoveries—low-binding plastic and small membrane surface area minimize sample loss Highlights: compared to filtration and resin systems • Easy to use—no knots or clamps required; just inject sample into cassette to begin dialysis (Figure 8) • One-step protocol—pipette sample into the Slide-A-Lyzer MINI Device and place in tube containing the dialysis • Fast dialysis—flat cassette chamber with two buffer; no laborious assembly, device preparation or membranes provides high surface-area-to-volume ratio expensive equipment is required that maximizes diffusion rate compared to cylindrical dialysis tubing • 100% leak tested—innovative design does not permit “wicking” that can occur in homemade devices • High recovery—rectangular cassette design maximizes recovery of entire sample volume via any one of the four • Minimal dialysis buffer required—minimizes waste The Thermo Scientific Slide-A-Lyzer MINI Dialysis Devices The original Thermo Scientific Slide-A-Lyzer Dialysis corner injection ports have a unique cup-like design and are available in 0.1, 0.5 The 0.1 mL devices can be placed into a foam float during Cassettes facilitate rapid and effective dialysis for sample • Four cassette sizes—select the cassette that best suits and 2 mL capacities. Slide-A-Lyzer MINI Dialysis Devices dialysis (Figure 6), and are available in MWCOs of 2K, 3.5K, volumes from 100 μL to 30 mL. The cassette design the sample volume allow easy sample addition and removal using a standard 7K, 10K, or 20K. The 0.5 mL and 2 mL sizes, which are maximizes surface area to sample volume ratio and provides laboratory pipette and can be used for single or arrays of integrated into 15 mL and 50 mL capped conical tubes, excellent sample recovery. Unlike standard flat tubing, these • Sterile option—gamma-irradiated 10K MWCO cassettes samples. The self-contained, single-use devices require no respectively, are available in 3.5K, 10K and 20K MWCO. The innovative cassettes do not require knots or clips that can are available for applications requiring sterilized conditions syringes, centrifuge, beakers, or laborious steps. Using the tubes serve as dialysis reservoirs for easy and self-contained lead to leaking and sample loss, resulting in more complete Slide-A-Lyzer MINI Dialysis Devices, low molecular weight dialysis (Figure 7). sample recovery.

1. Apply sample with a pipette. 2. Place the Slide-A-Lyzer MINI 3. Insert the float into the beaker 4. Recover sample. Dialysis Device into the float. containing the dialysate. Figure 6. Sample dialysis using a 0.1 mL Thermo Scientific Slide-A-Lyzer MINI Dialysis Device. The required float is sold separately.

Sample 1. Insert syringe needle through the 2. Attach a float buoy and dialyze. 3. Insert empty syringe needle at a gasket via one of the corner ports. (Buoys also serve as convenient second corner port. Inject air to expand the cassette chamber, then Buffer Inject the sample, withdraw the bench-top stands for the excess air and remove the syringe. cassettes.) withdraw the dialyzed sample.

Figure 8. Thermo Scientific Slide-A-Lyzer Dialysis Cassette procedure summary.

1. Add sample into 2. Place the device into the 3. Shake gently on 4. Replace dialysis buffer after 5. Remove the device from the device. conical tube containing the an orbital shaker. 2–3 hours and shake for an the conical tube and recover buffer and cap the tube. additional 2–4 hours or overnight. the sample. Figure 7. Sample dialysis with a 0.5 mL or 2 mL Thermo Scientific Slide-A-Lyzer MINI Dialysis Device. 10 11 Protein dialysis using regenerated cellulose membranes Protein clean-up technical handbook

Thermo Scientific Slide-A-Lyzer G2 Highlights: Thermo Scientific Slide-A-Lyzer The flasks are manufactured using clean-room conditions. Dialysis Cassettes • Easy loading—pipette-accessible for easy sample Dialysis Flasks The flasks are constructed from two sheets of low-protein loading and retrieval binding, regenerated cellulose membranes to help ensure maximum sample recovery and purity and contain up to Maximum convenience for high- • Self-floating—integrated air chambers eliminate the need Secure and easy to handle tool for 85% less plastic per volume compared to cassettes. performance dialysis for float buoys large-volume dialysis (150–250 mL) • Sturdy construction—enables high sample integrity Highlights: and protection • Easy to use—simply pipette or pour sample into flask, replace and tighten cap, and begin dialysis • Superior design—researched and tested to provide fast and consistent dialysis with maximum sample recovery • Fast dialysis—flat flask chamber with two membranes provides high surface-area-to-volume ratio, enabling • Multiple sizes—five cassette capacities to optimally dialysis of a 250 mL sample in as few as 8 hours match 0.25 to 70 mL sample volumes • High recovery—rectangular flask design maximizes • Versatile—ideal for removing low molecular weight recovery of entire sample volume via opening at top contaminants, performing buffer exchange and desalting of flask • Sterile option—gamma-irradiated 10K MWCO cassettes • Color-coded frames—easily identify membrane pore are available for applications requiring sterile conditions size (MWCO) based on the frame color Thermo Scientific™ Slide-A-Lyzer™ G2 Dialysis Cassettes Thermo Scientific Slide-A-Lyzer Dialysis Flasks facilitate provide maximum convenience, flexibility and performance The single-use, disposable Slide-A-Lyzer G2 Dialysis simple and effective removal of buffer salts and small Slide-A-Lyzer Dialysis Flasks eliminate the risk of sample loss for sample dialysis. These second-generation (G2) Cassettes are available in five MWCOs (2K, 3.5K, 7K, 10K, contaminants from proteins and other macromolecules in associated with handling long lengths of slippery dialysis Slide-A-Lyzer Dialysis Cassettes are freestanding, self- and 20K) and in five different sizes for dialyzing sample sample volumes up to 250 mL. Slide-A-Lyzer Dialysis Flasks tubing. No knots or clips are needed to seal the units. floating and pipette-loadable. Sample loading and removal volumes between 0.25 and 70 mL. The membrane is are available in four molecular weight cutoffs (MWCOs): Sample addition and removal are easily accomplished by are easily accomplished by using a serological pipette or composed of low-binding, regenerated cellulose, and the 2K, 3.5K, 10K, and 20K and are color-coded for easy pipetting or directly pouring the sample through the wide- hypodermic needle and syringe (Figure 9). The built-in air cassettes are manufactured using clean-room conditions. identification. With Slide-A-Lyzer Dialysis Flasks, typical low mouth opening at the top of the flask (Figure 10). A simple chamber provides sample buoyancy and vertical orientation Select sizes of 10K MWCO Slide-A-Lyzer G2 Dialysis molecular weight contaminant removal, buffer exchange screw cap easily and reliably seals the device. of the cassette during dialysis. Cassettes are also available in packages that have been and desalting can be accomplished in as few as 8 hours. gamma-irradiated to sterilize them. Gamma-irradiated Slide-A-Lyzer G2 Dialysis Cassettes are ideal for researchers culturing cells and microorganisms; purifying viruses, DNA, and RNA; or performing sample preparation for other applications requiring sterile conditions to minimize the risk of sample contamination.

Add sample Remove air Dialyze Figure 9. Thermo Scientific Slide-A-Lyzer G2 procedure summary.

Pipette

1. Install float ring. 2. Hydrate membrane. 3. Pour in sample. 4. Remove air. 5. Dialyze sample. 6. If needed, use 7. Pour out dialyzed syringe to remove sample. pressure. Syringe Figure 10. Easy sample loading and recovery with Thermo Scientific Slide-A-Lyzer Dialysis Flasks. Attach supplied float-ring and hydrate membrane for 2 minutes. Pour sample into device. Remove air and cap. Dialyze for 8 hours to overnight (replace buffer after 2 and 5 hours). Pour out sample to recover.

12 13 Protein dialysis using regenerated cellulose membranes Protein desalting using gel filtration resins Protein clean-up technical handbook

Thermo Scientific SnakeSkin Highlights: Dialysis Tubing • Convenient—ready-to-use, pre-wetted, pleated tube Protein desalting using gel • High recovery—up to 90% protein recovery Easier to use than traditional flat tubing • Speed—dialysis is generally completed in 4 to 6 hours filtration resins

• Stability—compatible with a variety of laboratory solutions, including acids, bases, hydrophobic solvents, In addition to the separation of macromolecules, SEC is also and alcohols Overview commonly used for the separation and removal of unwanted SnakeSkin Dialysis Tubing is composed of regenerated molecules from a macromolecule of interest (desalting), cellulose dialysis tubing and supplied as an open, pleated Size exclusion chromatography or exchange of the buffer for downstream applications (telescoped) tube. It is supplied in eight inch (20cm) sticks (buffer exchange). Applications for desalting include not containing 35 feet of dialysis tubing having a 16, 22 or (SEC) involves the chromatographic only the removal of salts, but also the removal of excess 35 mm circular internal diameter (I.D.). The hydrated biotin, crosslinkers, reactive dye, radioactive labels, or other SnakeSkin Dialysis Tubing holds ~2 to 10 mL of sample per separation of molecules of derivatization reagents from conjugation reactions. Buffer centimeter of length. Because SnakeSkin Dialysis Tubing is exchange is used to place a protein solution into a more made from the same type of regenerated cellulose as flat different dimensions, molecular appropriate buffer before subsequent applications such as Thermo Scientific™ SnakeSkin™ Dialysis Tubing is an easy- tubing, its dialysis performance matches that of conventional electrophoresis, ion exchange and affinity chromatography, and ready-to-use form of traditional dialysis membrane tubing. SnakeSkin Dialysis Tubing is available in three weight or size. Size exclusion or conjugation. tubing that allows desalting and buffer exchange for 10 to molecular weight cutoffs: 3.5K, 7K and 10K, with an internal 100 mL samples, and it does not require presoaking or diameter of 22 mm. The 3.5K and 10K MWCO membranes chromatographic resin usually How separation is achieved boiling prior to use. To use, simply pull out and cut off the are also available with internal diameters of 16 and 35 mm. Size exclusion chromatography applications for separating required length of tubing, fold over one end of tubing and consists of small, uncharged porous macromolecules based on subtle differences in size close with a dialysis clip, add sample at the open end, and particles with a range of pore sizes. typically use resins with large and varied pore sizes in long use a second clip to close the remaining end. chromatography columns. However, for buffer exchange and Molecules are separated based desalting applications, it is mainly the maximum effective pore size (exclusion limit or molecular weight cutoff (MWCO) on the relative abilities of molecules of the resin), which determines the size of molecules that can be separated. Molecules that are significantly smaller to penetrate into the pores. This than the MWCO penetrate into the pores of the resin, while molecules larger than the MWCO are unable to enter the technique is also commonly referred pores and remain together in the void volume of the column. By passing samples through a column resin bed with to as gel filtration or molecular sieve sufficient length and volume, macromolecules can be fully separated from small molecules that travel a greater distance Table 5. Dialysis tubing specifications and sample capacity. chromatography. Size exclusion though the pores of the resin bed. No significant separation Membrane Membrane Tubing Volume of molecules larger than the exclusion limit occurs (Figure 1). MWCO* (Da) Thickness Diameter (mL/cm tubing)† chromatography is used in research 3.5K 1.0 mil (25 μm) 16 mm I.D. ~2.0 mL and industrial applications for a wide 7K 1.2 mil (30 μm) 22 mm I.D. ~3.8 mL

10K 0.9 mil (23 μm) 35 mm I.D. ~9.6 mL range of applications ranging from

* Excludes membrane length used for tube closure † Membrane type: Regenerated cellulose the separation of proteins, DNA fragments and polymers. Learn more at thermofisher.com/dialysis

14 15 Protein desalting using gel filtration resins Protein clean-up technical handbook

In order for the desired macromolecules to remain in the void For dialysis applications, achieving a high-percentage Resin performance data gravity flow and require no chromatography system, allowing volume, resins with very small pore sizes must be utilized. sample recovery and molecule removal is generally for multiple sample processing simultaneously. However, For routine desalting and buffer exchange applications, straightforward with little optimization needed. For gel Optimized products for improved protein recovery due to the lack of a chase buffer, spin column methods have choosing a resin with a molecular weight cutoff between filtration applications it is important to select a column size and faster desalting historically suffered from sample loss, particularly at low 5 and 10 kDa is usually best. For other applications, and format that is suitable for your sample. To eliminate sample dilution and the collecting and protein concentrations, and the sample volume they could such as separating peptides from full-sized proteins, monitoring of fractions, centrifuge-column or plate-based be used with were limited. resins with larger exclusion limits may be necessary. The Gravity-flow, or drip, columns use head-pressure from a gel filtration, also referred to as spin desalting methods, macromolecular components are recovered in the buffer buffer chase to push the sample through the gel filtration are commonly used. Spin desalting is unique in that a Thermo Scientific Zeba desalting products contain a used to pre-equilibrate the gel-filtration matrix, while the matrix. The sample is loaded into the top of an upright centrifuge is used to first remove the resin’s void volume unique resin specifically designed to provide consistent small molecules can be collected in a later fraction volume or column and allowed to flow into the resin bed. The sample of liquid, followed by sample addition and centrifugation. performance over a wide range of protein concentrations left trapped in the resin. One important feature to note when is then chased through the column by adding additional After centrifugation the macromolecules in the sample have and sample sizes. High protein recovery can be achieved choosing a resin is that the small molecules targeted for buffer or water to the top of the column. During this process, moved through the column in approximately the same initial even for dilute protein samples. Two MWCOs (7K and removal must be several times smaller than the MWCO for small fractions are typically collected and tested for the volume, but the small molecules have been forced into the 40K MWCO) are available to optimize for larger or smaller proper separation. macromolecules of interest. As an alternative to fraction pores of the resin and replaced by the buffer that was used proteins and/or contaminants. Multiple formats can collection, a single fraction equal to the full exclusion volume to pre-equilibrate the gel-filtration matrix (Figure 2). Spin accommodate the different needs for sample volumes, Desalting vs. dialysis of the column is collected regardless of the sample volume. formats eliminate the need to wait for samples to emerge by automation and throughput. Dialysis is useful for many of the same desalting and This eliminates the time and monitoring associated with buffer exchange applications performed with gel filtration fraction collecting; however, this can result in significant chromatography as both methods are based on similar dilution of the sample depending on the sample volume. ranges of molecular weight cutoffs, but gel filtration is Gel filtration formats for smaller volumes include gravity-flow faster (a few minutes vs. hours for dialysis). An additional columns, chromatography cartridges, centrifuge columns advantage of gel filtration is the ability to remove and centrifuge plates. contaminants in a relatively small volume (or left on the column), an important feature when working with toxic or 1. Remove bottom 2. Centrifuge for 1–2 3. Apply equilibration buffer. 4. Apply sample. Centrifuge Recover desalted radioactive substances. Dialysis, on the other hand, is much plug. Place columns minutes at 1,000–1,500 × Centrifuge 1–2 minutes for 1–2 minutes at sample. in a collection tube. g to pack bed and at 1,000–1,500 x g. 1,000–1,500 × g. less dependent on sample size as related to device format. remove storage buffer. Discard flow through. Repeat 2–3 times.

Figure 2. Protocol for desalting with Thermo Scientific™ Zeba™ Spin Desalting Columns.

Figure 1. Passage of a protein sample through Table 1. Thermo Scientific™ Zeba™ resin selection guide by protein recovery and small molecule removal. a column of porous resin facilitates buffer exchange 7K 40K and desalting. Small molecules in the original Size Recovery Removal Recovery Removal sample (red) enter the bead Peptide/Protein <7 kDa NR NR pores, thereby taking a longer and slower path through Protein 7–13 kDa ++ + the column than the protein (yellow). As a result the protein Protein 14–20 kDa +++ ++ separates from the original buffer salts and exchanges Protein 20–150 kDa +++ +++ into the column buffer. Molecule <500 Da +++ +++

Molecule 600–1,200 Da ++ +++

Molecule 1,200–1,500 Da + ++

Molecule >1,500–2,000 Da NR +

NR = Not Recommended + = Good ++ = Better +++ = Recommended

16 17 Protein desalting using gel filtration resins Protein clean-up technical handbook

Table 2. Thermo Scientific Zeba Spin Desalting Columns provide load volume may exceed recommended ranges (see below). There were no Protein recovery compared to other vendors exceptional protein recovery over a wider range of sample significant differences in salt concentration between any vendor when used concentrations and volumes compared to alternative products. within the recommended ranges. Protein concentration was determined Desalting columns were equilibrated with a final buffer containing 25 mM using the Thermo Scientific™ Pierce™ BCA Protein Assay (Cat. No. PI23227). Tris, 25 mM NaCl at pH 7.5. Samples were diluted in 25 mM Tris, 500 mM Recoveries with lysate are typically slightly lower than seen with individual 1.5 mL 2.5 mL 3.5 mL 40 µL 80 µL 120 µL NaCl at pH 7.5 and then desalted into the final buffer. All samples were proteins due to the loss of small molecular weight compounds and lysis processed according to the manufacturers’ recommended instructions, but contaminants that can influence protein assay results. 0.04 mg/mL BSA 7K Zeba Bio-Rad 7K Zeba Spin GE Micro Spin GE Sample Columns, SpinTrap Bio-Spin Sample Columns, PD-10 0.2 mg/mL volume 0.5 mL G-25 6 Columns volume 10 mL Columns BSA

Resin bed volume 550 µL 500 µL 700 µL 10 mL 8.3 mL 8.3 mL 1.0 mg/mL BSA Method Spin Spin Spin Spin Spin Drip

% Protein recovery % Protein recovery Thermo Scientific Thermo Scientific Thermo Scientific BSA 0.04 mg/mL 40 µL 70–80% <10% <10% 1.5 mL 75–90% 20–30% 40–50%*** Load Spin GE GE Gravity Spin GE GE Gravity Spin GE GE Gravity

80 µL 80–95% <10% <10% 2.5 mL 75–90% 30–40% 45–55%*** Figure 3. Thermo Scientific Zeba Spin Desalting Columns provide a 120 µL >90% <10% <20% 3.5 mL 75–90% 60 –70%* 55– 65%* high protein recovery while providing minimal sample dilution over a wider range of sample concentrations and volumes compared to BSA 0.2 mg/mL 40 µL 70–80% <15% 25–35% 1.5 mL >90% 60–70% >90%*** alternative products. Zeba Spin Desalting Columns, 10 mL (7K MWCO) (Cat. No. PI89893) and GE PD-10 Columns were used to desalt 1.5, 2.5 80 µL 80–95% 30–45% 45–60% 2.5 mL >90% 70–80% 85–95%*** Load Control Thermo Scientific GE Bio-Rad Thermo Scientific GE Bio-Rad Thermo Scientific GE Bio-Rad and 3.5 mL BSA samples at a concentration of 0.04, 0.2 and 1 mg/mL. 120 µL >90% 55–70% 65–80% 3.5 mL >90% 75–85%** 75–85%** Desalting was performed according to the manufacturers’ recommended Figure 4. Better performance with Thermo Scientific Zeba Spin protocols, both the spin and gravity protocols were used for the GE PD-10. Desalting Columns. Zeba Spin Desalting Columns provide a high protein BSA 1 mg/mL 40 µL 80–95% 45–60% 70–80 1.5 mL >90% 80–90% >90%*** Protein recovery was analyzed by SDS-PAGE. For each electrophoresis recovery while providing minimal sample dilution over a wider range of gel, an aliquot of starting sample equal to 1 µg of BSA was loaded in sample concentrations and volumes compared to alternative products. 80 µL >90% 60–75% 80–90 2.5 mL >90% 80 –90%* >90%*** Lane 1 as the Load Control; all other desalted samples were loaded in Zeba Spin Desalting Columns, 0.5 mL (7K MWCO) (Cat. No. PI89882), 120 µL >90% 75–90% >90% 3.5 mL >90% 80 –90%* 85–95%* the gel at the same volume as the Load Control. Differences in intensity GE SpinTrap G-25 and Bio-Rad Micro Bio-Spin 6 spin columns were used between lanes are a combination of protein recovery and sample dilution to desalt 40, 80 and 120 µL samples of HeLa lysate at a concentration HeLa lysate 0.2 mg/mL** 40 µL 45–55% <10% <15% 1.5 mL 70–85% 55–65% >90%*** caused by desalting. The largest differences in recovery and concentration of 0.2 and 1 mg/mL. Desalting was performed according to the were noticed in the highlighted area. manufacturers’ recommended protocols. Protein recovery was analyzed 80 µL 60–70% 15–25% 30–40% 2.5 mL 70–85% 65–75% 80–95%*** by SDS-PAGE. For each electrophoresis gel, an aliquot of starting sample 120 µL 70–80% 45–55% 50–60% 3.5 mL 80–95% 70 –80%* 75–85%* equal to 1 µg of HeLa lysate was loaded in Lane 1 as the Load Control; all Table 3. Comparison of protein recovery, from the 7K MWCO other desalted samples were loaded in the gel at the same volume as the HeLa lysate 1 mg/mL** 40 µL 65–75% 45–55% 45–55% 1.5 mL 80–95% 80–90% >90%*** and 40K MWCO, 0.5 mL Thermo Scientific Zeba Spin Desalting Load Control. Differences in intensity between lanes are a combination of Columns. protein recovery and sample dilution caused by desalting. 80 µL 75–85% 60–70% 70–80% 2.5 mL 80–95% 80–95% >90%*** Typical % recovered from 100 µL sample 120 µL 80–90% 70–80% 80–90% 3.5 mL 85–95% 80 –95%* 85–95%* Table 4. Comparison of small molecule removal, for the 7K MWCO 40K * Note: The recommended sample volumes for the columns tested above would result in insufficient salt removal. They are included only to help demonstrate the effect of a small volume on protein recovery. and 40K MWCO, 0.5 mL Thermo Scientific Zeba Spin Desalting Concentration Size of 7K Zeba Zeba Columns. **Note: Recoveries with lysate are typically slightly lower than those seen with individual proteins due to the loss of small molecular weight compounds and lysis contaminants that can influence protein assay results. Recovery of loaded molecule Column Column Typical % removed ***Note: Sample diluted during processing and recovered in 3.5 mL final volume. Protein recovery determined on total final volume. Ubiquitin 0.5 mg/mL 8.7 kDa 75% 60% from 100 µL sample 40K α-Lactalbumin 1.0 mg/mL 14.1 kDa 85% 75% Concentration Size of 7K Zeba Zeba Removal of loaded molecule Column Column Soybean Trypsin 0.5 mg/mL 20.1 kDa 85% 65% NaCl 1 M 58.44 Da >99% >99% Inhibitor Dithiothreitol 0.5 M 154 Da 99% 99% Carbonic Sulfo-NHS- 0.5 mg/mL 29 kDa 90% 75% 0.27 mM 557 Da 85% 85% Anhydrase LC-Biotin Ovalbumin 0.5 mg/mL 44 kDa 90% 85% Dy549 Dye 0.2 mM 1,026 Da 75% 90% Bacitracin 0.5 mg/mL 1,200 Da 75% 95% Bovine Serum 0.5 mg/mL 66 kDa >90% >90% Albumin Vitamin B12 0.5 mg/mL 1,386 Da 85% 95%

Human IgG 0.5 mg/mL 150 kDa >90% >90%

18 19 Protein desalting using gel filtration resins Protein clean-up technical handbook

A. Thermo Scientific Pierce Cartridge Selecting the right format for sample processing (Thermo Scientific Zeba Resin, 7K MWCO)

mAU Figure 5. Efficient salt removal and protein recovery with Thermo Scientific Zeba Desalting Chromatography Cartridge. Bovine serum Thermo Scientific Zeba desalting products contain Unlike the limited offerings of other suppliers, Zeba Spin 600 albumin (1 mg) in 1 M NaCl was applied to 5 mL Zeba Desalting Cartridge proprietary high-performance resins with exceptional Desalting Columns are available in a wide range of formats (Cat. No. 89935) (A) at a flow rate of 5 mL/minute. Cartridge profile shows 500 desalting and protein-recovery characteristics compared to match a variety of applications (Table 5). Our broad isocratic elution of BSA (blue) and NaCl detected by conductivity (brown). 400 Greater than 95% of the BSA was recovered and more than 95% of the to other commercially available spin desalting media. Even portfolio of high-performance devices for desalting and

300 salt was removed. Results for the Pierce Cartridge (A) were essentially very dilute protein samples can be successfully processed buffer exchange provide easy handling, rapid processing identical to those obtained with the more expensive GE Healthcare (B) with high levels of protein recovery and greater than 95% and exceptional recovery for sample volumes between 200 and Bio-Rad (C) products. Conductivity (brown) Conductivity retention (removal) of salts and other small molecules. Zeba 2- and 4,000 μL. In addition, two size-exclusion resin options 100 Absorbance (280 nm) (blue) desalting products rapidly process sample volumes ranging (7K and 40K MWCO) are available. The 7K Zeba Desalting 0 from 10 μL to 4 mL. The combination of the unique resin and Resin is recommended for removing molecules <800 Da 0.0 2.0 4.0 6.0 8.0 10.0 Chromatography progress (mL) easy-to-use column, plate and cartridge formats help ensure from macro molecules greater than 7 kDa. The 40K Zeba maximum protein recovery in minimum time. Desalting Resin is recommended for removing molecules <1,500 Da from macro molecule greater than 30–40 kDa. B. GE Healthcare Cartridge (HiTrap Column with Sephadex G-25 Resin) Highlights: Salt removal is typically 95–100%. Examples of protein-

mAU • High performance—proprietary resin enables excellent specific recovery, contaminant removal data, and 700 protein recovery and efficient contaminant removal benchmarks versus similar products from other suppliers 600 are shown on pages 17–20. 500 • Flexible—available in spin columns, filter spin plates,

400 and cartridges for a range of needs 300 • Fast—no fraction screening or waiting for protein to 200 emerge by gravity flow

Conductivity (brown) Conductivity 100

Absorbance (280 nm) (blue) • Economical—cost-effective products that offer 0 0.0 2.0 4.0 6.0 8.0 10.0 great performance Chromatography progress (mL)

Table 5. Thermo Scientific Zeba desalting products selection guide by format C. Bio-Rad Cartridge (Bio-Scale Mini Cartridge with Bio-Gel P-6 Resin) Zeba Spin Zeba 96-well Zeba Chromatography Desalting Columns Spin Plates Columns mAU

600

500

400

300

200 Sample volume range 2 μL–4 mL 20–100 μL 15–250 μL or 100 –1,500 μL Conductivity (brown) Conductivity 100

Absorbance (280 nm) (blue) Bed volume range 75 μL–10 mL 550 μL 1 mL or 5 mL 0 0.0 2.0 4.0 6.0 8.0 10.0 Number of devices with different 5 1 2 Chromatography progress (mL) volume capacities

Molecular weight cutoffs 7K, 40K 7K, 40K 7K (MWCOs)

Automation compatible? No Yes No

FPLC compatible? No No Yes

20 21 Protein desalting using gel filtration resins Protein clean-up technical handbook

Zeba Spin Desalting Columns Zeba Spin Desalting Columns are made of low protein Table 7. Comparison of recommended sample volume capacities of common spin desalting products. binding polypropylene and are compatible with a wide range 0 mL 0.01 mL 0.1 mL 0.5 mL 1 mL 2 mL 3 mL 4 mL of standard laboratory instruments and consumables. The Zeba Micro 0.5 mL 2 mL Zeba Zeba Spin Desalting Columns and Plates are designed to be Thermo Scientific Spin Column Zeba Spin Column Spin Column 10 mL Zeba Spin Column compatible with most swinging-bucket or fixed-angle bench Zeba Spin Desalting Products or floor model centrifuges; however, proper head clearance 5 mL Zeba Spin Column should be verified before use. PD SpinTrap G-25 Column GE Healthcare Products PD MiniTrap PD MidTrap PD-10 G-25 Column G-25 Column Desalting Columns Micro Bio-Spin 6 Column Bio-Rad Products Bio-Spin 6 Column

Table 6. Thermo Scientific Zeba desalting products selection guide by format, recommended sample volume, and column schematics.

Zeba Spin Desalting Plates The pre-packed Thermo Scientific™ Zeba™ 96-well Spin Desalting Plates do not require resin hydration or dispensing and provide the same high protein recovery as Zeba Spin Desalting Columns. Each Zeba Spin Desalting Plate can process up to 96 samples (20 to 100 μL) in as Format Micro spin column 0.5 mL spin column 2 mL spin column 5 mL spin column 10 mL spin column few as 10 to 20 minutes. A collection plate is provided with Resin bed 75 µL 0.5 mL 2 mL 5 mL 10 mL each filter plate. Sample volume 2–12 µL 30–130 µL 200–700 µL 500–2,000 µL 700–4,000 µL (7K MWCO) Sample volume 5–14 µL 70–200 µL 200–900 µL 300–2,000 µL 1,000–4,000 µL (40K MWCO) Thermo Scientific Zeba Spin Table 8. Common specifications of Thermo Scientific Zeba Thermo Scientific Zeba Micro Spin Thermo Scientific Zeba Spin Thermo Scientific Zeba Spin Desalting Columns, 0.5 mL Thermo Scientific Zeba Spin Desalting Columns, 10 mL Desalting Columns, 75μL Desalting Columns, 2mL Desalting Columns, 5 mL 96-well filter plates. Total column capacity = 0.4 mL Total column capacity = 0.8 mL Total column capacity = 5.5 mL Total column capacity = 8.5 mL Total column capacity = 23 mL Plate dimensions (l x w x h)† 127.76 x 85.5 x 45 mm ± 0.25 mm (resin bed = 0.1 mL; reservoir = 0.3 mL) Well depth 27.11 ± 0.10 mm 13 mm 21.5 mm 21.5 mm 13 mm 34 mm Well diameter 7 ± 0.10 mm

8 mm 3.5 mL Well off-set 9 mm 8 mm 3.2 mL 13 mL 23 mm reservoir 23 mm reservoir 27 mm reservoir volume 6 mm volume volume Well volume 800 µL

5 mL 10 mLml Plate material polypropylene 37 mm 9 mLml 39 mm 18 mm 25 mm 4 mL 29 mm 8 mLml Filter material polyethylene 23 mm 7 mLml 112 mm 2.3 mL 105 mm 10 mL 100 mm 5 mL 3 mL 6 mLml column column 54 mm column 59 mm 5 mLml Filter pore size 20 µm volume volume 62 mm volume 4 mLml 2 mL 7 mm 3 mLml Collection plate maximum volume 150 µL 2 mLml 6 mm (twist- 1 mL 1 mLml off tab) Max centrifuge speed up to 1,000 x g 9 mm 9 mm 4 mm 9 mm twist-off Suggested balance plate use Cat. No. PI45205 9 mm tab 14 mm Total height with twist-off 14 mm twist-off top and bottom tab tab 14 mm † Plate height includes collection plate for total stack height. caps in place = 44 mm 15 mm (inner) 16.5 mm (outer) 7.5 mm (inner) 10.5 mm (inner) 9 mm (outer) 12 mm (outer)

22 23 Protein desalting using gel filtration resins Detergent removal using chromatography resins Protein clean-up technical handbook

Zeba Desalting Chromatography manually or by automated liquid chromatography (LC) Cartridges systems. The cartridges attach directly to the ÄKTA™ FPLC™ Detergent removal using system from GE Healthcare or other FPLC systems without additional connectors. Cartridge products include an Thermo Scientific™ Zeba™ Desalting Chromatography accessory pack of tubing fittings and Luer-Lok™ fittings that chromatography resins Cartridges are available pre-packed in 1 mL and 5 mL provide compatibility with the other popular LC systems sizes. They can be regenerated for multiple uses and and manual syringe processing. They are available only in efficiently process samples from 50 to 1,500 μL. Zeba the 7K MWCO resin. molecules will be in micelles that are too large to diffuse Desalting Chromatography Cartridges can be processed Overview through the pores of the dialysis membrane; only excess monomer can be dialyzed. Ion exchange chromatography using appropriate conditions to selectively bind and elute Table 9. Thermo Scientific Zeba Desalting Chromatography Detergents are a class of Cartridge properties. Recommended and maximum flow rates are the proteins of interest is another effective way to remove general; values differ slightly for individual products. molecules whose unique detergents. For peptides, ion exchange can be used to bind Feature 1 mL Cartridge 5 mL Cartridge and remove selective detergents, but only if they are anionic Dimensions 0.7 x 2.7 cm 1.3 x 3.8 cm properties enable manipulation or cationic detergents, such as SDS. Sucrose density Desalting flow rate 0.2 to 1 mL/minute 1 to 5 mL/minute gradient separation also can be used. However, all these (Maximum) (3 mL/minute) (8 mL/minute) (disruption or formation) of methods can be somewhat labor- and/or time-intensive or Affinity flow rate 0.1 to 1 mL/minute 0.5 to 2 mL/minute detergent-specific. (Maximum) (4 mL/minute) (5 mL/minute) hydrophobic and hydrophilic 0.3 MPa 0.3 MPa Maximum pressure (43 psi or 3 bar) (43 psi or 3 bar) interactions among molecules in Improved tools to quickly and efficiently Cartridge material polypropylene polypropylene remove detergents ™ ™ Frit material polyethylene polyethylene The Thermo Scientific Pierce detergent removal products biological samples. In life science contain a proprietary resin that specifically bind a wide variety of detergents and surfactants that are commonly Thermo Scientific Zeba Chromatography Cartridges applications, detergents are used used in protein extraction and biological sample preparation. The spin column format provides a convenient and rapid Port diameter: 4.2 mm for cell lysis, protein solubilization 10–32 threaded coned port method for removing interfering detergents from protein and peptide solutions before downstream analysis by MS and

17.5 mm 17.5 and denaturation, or to reduce 20 mm other techniques. Samples can be processed in as little as 12.5 mm 13.5 mm background in certain applications. 15 minutes. 5 mL 89 mm 1 mL

14 mm 75 mm 8.5 mm The detergents and surfactants used to prepare protein and peptide samples can interfere with analysis by ELISA,

4 mm isoelectric focusing and mass spectrometry (MS). Removing 4 mm detergents from peptide samples is especially challenging and critical for MS analysis because even low concentrations of detergents will contaminate instruments and interfere with column binding, elution and peptide ionization. Learn more at thermofisher.com/desalting Detergent removal can be attempted in a number of ways. Acetone or acid precipitation can be used for proteins (not peptides) and generally has poor recovery. Dialysis is effective for removal of detergents that have very high Critical Micelle Concentration (CMC) and/or small aggregation numbers, such the n-octyl glucoside formulations. Detergents with low CMCs and large aggregation numbers cannot be dialyzed because most of the detergent

24 25 Detergent removal using chromatography resins Protein clean-up technical handbook

Resin performance data LC/MS analysis of digested peptide

43.39 43.39 47.20 47.20 100 35.61 100 35.61 100 100 100 100 35.61 34.01 37.69 34.01 37.69 43.39 44.18 44.18 40.37 40.37 100 47.20 100 35.5036.14 35.5036.14 100 100 100 100 100 45.67 45.67 34.0135.50 37.69 44.18 100 40.37 95 45.67 95 36.14 95 95 95 Detergent removal efficiency and protein recovery 100 95 95 95 95 95 95 95 95 90 90 90 90 95 95 90 90 43.83 43.83 90 36.21 36.21 90 90 90 90 90 90 43.83 44.06 44.06 85 36.2133.86 36.4985 37.90 44.06 33.86 36.49 37.90 85 85 90 90 85 85 33.86 38.82 43.88 38.82 43.88 85 85 39.70 39.70 85 42.89 42.89 85 36.49 37.90 37.15 39.67 37.15 39.67 85 85 85 38.82 43.88 43.37 85 39.70 80 42.89 80 37.15 39.67 80 40.55 42.97 44.26 47.3240.55 42.9743.3744.26 47.32 80 80 85 32.13 32.13 44.27 44.27 80 30.4730.71 40.55 30.4743.37 41.73 41.73 32.13 80 80 44.27 41.42 41.42 80 80 30.47 34.30 41.7342.9730.71 44.26 42.2734.3047.32 45.78 42.27 45.78 80 80 80 33.01 33.01 30.71 45.78 44.30 46.22 47.74 44.30 46.22 47.74 80 80 33.01 41.42 39.03 42.48 39.03 42.48 75 75 75 34.30 75 42.27 47.74 75 75 39.03 41.08 43.56 41.08 43.56 75 44.30 46.22 75 75 75 75 42.48 75 31.47 31.47 44.43 44.43 75 ™ ™ 41.08 43.56 70 31.63 70 31.6344.43 44.63 46.57 44.63 46.57 75 75 31.84 31.84 70 70 31.4731.63 33.69 33.69 70 70 70 30.03 70 38.7030.03 42.57 38.70 42.57 70 70 33.69 44.63 46.57 70 45.46 45.46 70 70 31.84 36.42 42.57 36.42 50.06 50.06 32.73 32.73 Table 2. Results using standard Thermo Scientific Pierce 70 43.50 43.50 70 30.03 36.42 33.6238.70 33.62 45.4050.0646.58 48.95 45.4046.58 48.95 65 65 32.7365 65 65 65 45.46 33.62 45.40 48.95 50.53 52.10 50.53 52.10 65 48.54 65 65 43.50 65 30.60 33.9234.25 6538.3830.60 33.9246.5834.25 38.3847.39 54.4747.3965 54.47 40.26 40.26 65 48.54 46.93 46.93 65 33.92 38.38 36.65 47.39 36.65 50.53 52.10 40.26 60 60 48.54 65 30.60 34.2531.81 36.65 36.09 31.81 36.09 46.52 54.47 46.52 60 60 60 60 46.93 60 36.09 34.55 37.3860 46.5234.55 37.38 48.83 51.75 48.83 51.75 49.46 49.46 60 60 60 31.81 51.75 54.31 60 54.31 49.46 60 60 34.55 37.38 48.83 48.08 51.37 53.63 48.08 51.37 53.63 55 55 60 48.08 51.37 54.31 55 55 53.28 55 53.28 55 55 55 55 55 53.6349.77 55 49.77 55 49.3249.85 55 49.3249.85 46.23 46.23 53.28 50.94 50.94 Detergent Removal Spin Column, 0.5 mL. Detergent removal 55 46.16 46.16 55 49.77 50 50 46.74 46.74 50 50 49.3249.85 50 50 39.59 40.87 39.59 40.87 50 50 46.23 50.94 50.2751.43 50.2751.43 50 39.59 50 46.16 50 46.74 50 50 50 40.87 47.56 47.56 50 37.08 37.08 50.2751.43 47.56 45 45 45 45 51.9452.53 4551.9452.53 45 45 45 45 45 45 37.08 45 51.9452.53 45 45 54.59 54.59 ™ ™ 45 40 40 40 40 54.59 39.68 42.69 39.68 42.69 41.47 41.47 40 40 40 39.6836.7940 42.69 36.79 40 40 40 38.92 38.92 40 40 40 41.4737.92 37.92 40 40.83 40.83 36.79 Table 1. Results using Thermo Scientific HiPPR Detergent efficiency and protein recovery. BSA sample (25–200 µL) + detergent 37.92 35 38.92 40.8335 35 35 35 35 35 35 35 3538.00 38.00 35 35 35 43.83 43.83 35 35 43.83 38.00 47.15 47.15 30 30 30 30 30 30 30 30 47.15 30 30 30 30 38.35 38.35 30 44.77 44.77 30 37.54 37.54 30 38.35 40.22 40.22 25 37.54 25 25 25 25 40.2225 39.57 46.5539.57 46.55 25 25 40.0244.77 40.02 25 25 25 25 46.55 49.55 49.55 25 40.02 25 35.88 35.88 25 39.57 38.15 38.1549.55 Each column and plate well contained ~550 μL of in 0.15 M NaCl, 0.05% sodium azide was mixed with equal volume of 39.37 39.37 20 20 20 35.88 20 20 20 20 38.15 20 Removal Resin. 42.43 20 20 44.82 50.0242.43 44.82 50.02 20 20 40.34 40.34 39.37 42.43 44.82 20 20 20 50.02 39.51 39.51 40.3437.24 37.24 36.81 36.8145.54 45.54 15 15 15 15 15 15 15 35.62 15 35.62 15 15 15 39.5136.18 38.67 36.18 38.67 15 37.24 15 36.81 45.54 15 38.67 41.78 41.78 15 35.62 38.18 38.18 10 36.18 1041.78 42.25 45.42 42.25 45.42 10 10 10 10 45.42 48.23 48.23 10 10 10 10 41.35 41.35 38.18 49.23 52.3252.84 52.84 10 34.33 42.25 34.33 10 41.35 38.99 45.25 38.99 45.25 10 34.52 34.52 51.48 54.0154.89 49.2351.48 52.32 54.0154.89 10 48.2344.66 44.66 10 34.39 35.10 34.39 35.10 49.23 52.3252.8448.72 54.89 48.72 5 34.33 33.77 5 44.6633.77 50.78 50.78 5 5 38.99 45.25 44.25 48.15 44.25 48.15 detergent-removal resin slurry and 0.1 mL of sample. Similar results detergent removal resin (2x volume for CHAPS removal) and processed 5 34.52 5 51.48 54.01 53.92 53.92 5 5 32.2032.31 34.85 32.2032.31 34.85 49.84 49.84 5 5 48.15 54.57 54.57 34.39 35.10 33.88 35.64 33.88 35.6448.72 5 5 33.7730.4534.85 30.45 50.78 51.1852.44 54.23 51.1852.44 54.235 44.25 5 30.4731.5932.09 30.4731.5932.09 53.92 32.2032.31 49.84 54.57 5 32.6133.06 34.72 32.6133.06 34.72 51.03 52.68 51.03 52.68 31.5932.09 33.88 35.64 0 0 30.45 0 0 51.1852.44 54.23 0 0 31.93 31.93 49.30 53.0654.86 49.30 53.0654.86 30.47 0 0 31.9332.6133.060 34.72 0 49.30 51.03 52.6853.06 0 0 0 30 31 32 33 34 35 36 37 3038 3139 3240 3341 3442 3543 3644 3745 3846 3947 4048 4149 4250 4351 4452 4553 4654 4755 48 49 50 51 52 530 54 55 54.86 31 32 33 34 35 36 37 38 3931 4032 4133 4234 4335 4436 4537 4638 4739 4840 4941 5042 5143 5244 5345 5446 47 48 49 50 51 52 53 54 30 31 32 33 34 35 36 37 383039 3140 3241 3342 3443 3544 3645 3746 3847 3948 4049 4150 4251 4352 4453 4554 4655 47 48 49 50 51 52 53 54 55 30 31 32 33 34 35 36 3730 38 31 39 32 40 33 41 34 42 35 43 36 44 37 45 38 46 39 47 40 48 41 49 42 50 43 51 44 52 45 53 46 54 47 55 0 48 49 50 51 5230 53 31 54 32 55 33 34 35 36 3730 38 31 39 32 40 33 41 34 42 35 43 36 44 37 45 38 46 39 47 40 48 41 49 42 50 43 51 44 52 45 53 46 54 47 55 48 49 50 51 52 53 54 55 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Time 47 (min) 48 49 50 51 52 53 54Time (min) Time (min) Time (min) Time (min) Time (min) Time (min) Time (min) 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min) Time (min) Time (min) were obtained with both process formats. as shown in the protocol (page 29). Time (min) Time (min) Time (min) Time (min) No detergent,No detergent, unprocessedNo unprocessed detergent, unprocessed + 1% lauryl+ 1% maltoside,lauryl maltoside,+ 1% unprocessed lauryl unprocessed maltoside, unprocessedLauryl Laurylmaltoside, maltoside, processedLauryl processed maltoside, processed + 1% NP-40,+ 1% NP-40, unprocessed+ unprocessed 1% NP-40, unprocessed NP-40,NP-40, processed processedNP-40, processed Detergent Detergent BSA Sample Protein Detergent Protein 536.07 536.07 100 100 590.54 590.54 518.98 518.98 546.43 546.43 536.07 100 100 518.99 518.99 100 100 100 100 100 590.54 546.53 546.53 518.99100 536.08 100 536.08 518.98 100 546.43 519.00 519.00 100 100 100 519.0095 95 546.53 536.08 volume quantity removal recovery 95 502.41 95 502.41 95 95 95 95 95 95 95 502.41 95 95 95 590.47 590.47 90 90 95 Process concentration removal recovery 90 590.47 90 90 90 90 90 90 90 90 90 90 90 85 85 507.58 507.58 90 85 85 507.5885 634.51 85 634.51 85 85 85 85 85 85 820.75 820.75 85 85 634.51 85 820.75 † 80 80 80 80 Detergent (µL) (µg) (%) (%) 80 80 80 80 80 80 80 80 80 80 634.47 634.47 75 75 80 format Detergent (%) (%) (%) 75 75 75 634.47458.39 75 458.39 75 75 463.59 75 463.59 75 75 75 458.39 75 463.59 75 445.07 445.07 70 70 75 445.07 70 70 70 70 70 70 70 70 70 536.08 536.08 70 70 70 70 536.08 65 65 65 65 65 65 65 65 639.60 65 639.60 65 65 65 65 639.60 65 678.51 678.51 65 60 60 60 60 60 60 60 60 60 678.51 60 60 863.40 863.40 60 60 678.50 678.50 445.08 445.08 60 419.56 419.56 60 678.50 445.08 55 55 863.40 55 55 1176.98 1176.98 55 55 55 419.56 55 55 55 55 55 55 55 55 1176.98 50 50 683.61 683.61 445.08 445.08 50 50 50 50 50 50 50 50 SDS (1%) 25 0.375 >99 98 50 50 50 683.61 445.08 874.63 874.63 50 50 419.45 419.45 45 45 874.63 45 45 419.45 45 45 45 45 45 45 Sodium 45 45 0.5 mL 45 45 45 40 40 40 40 40 722.5240 722.52 40 40 40 40 40 40 610.13 863.39 610.13 863.39 40 40 722.52 610.13 722.54 722.54 40 610.14637.30 610.14637.30 5 99 100 592.87 592.87 35 35 35 863.3935 35 722.54 727.61 727.61 637.30 35 35 35 35 610.14 35 592.87 35 35 727.61 701.11 701.11 35 410.28 410.28 35 35 701.11 410.28 462.01 462.01 30 30 30 30 30 30 462.01 30 610.12 30 419.23462.01 419.23462.01 30 30 deoxycholate 610.12 30 763.04 763.04 30 419.23 30 Spin 30 30 462.01 610.12 763.04 25 474.44 708.69 474.44 708.69 25 25 785.41 785.41 25 25 25 25 25 474.44 25 419.23 708.69582.81 25 419.23 582.81 25 25 700.88 700.88 419.23 25 785.41 784.78 784.78 25 766.53 766.53 25 582.81 820.67 820.67 20 20 20 700.88 708.6520 875.02 708.65 875.02 20 766.53 20 740.73 740.73 20 20 20 708.65784.78666.77875.02 666.77 20 740.73 20 50 0.75 >99 97 20 820.67 20 782.97 782.97 582.77 820.81 582.77 820.81 20 20 481.06 481.06 15 15 15582.77 666.77 637.38820.81 15 637.38 785.19 899.45 785.19 899.45 481.06 15 15 782.97 637.38 740.81 740.81 15 15 15 15 956.43 956.43 666.82 740.83 666.82 740.83 15 15 887.45 997.59 887.45 997.59 785.19 899.45956.43 997.61 997.61 Column 15 740.81 997.59 917.54 15 810.55 810.55 15 941.34 941.34 666.82 740.83 10 10 10 887.4510 917.54 997.61 10 584.15 708.6110 875.20584.15 708.61 875.20 508.04 917.54 508.04 954.76 1171.82 954.76 1171.82 10 810.55 10 10 10941.34 584.15 653.83875.20 863.18 653.83 863.18 10 815.69 815.69 10 508.04 10 10 708.61 815.69 854.60 854.60 954.76 1171.82 1182.15 1182.15 859.70 859.70 10 1172.19 1172.19 653.83 863.18 941.03997.55 1192.68 941.03997.55 1192.68 5 5 5 5 1048.571112.57 1250.06 1048.571112.57 1250.06 5 1030.95 1229.89 1030.95 1229.89 5 5 1192.68 5 854.60 902.80 1037.79 902.80 1037.79 1048.57 1182.151250.06 1286.761356.781389.82 1513.35 1286.761356.781389.82 1513.35 859.705 5 5 1030.95 1172.19 1112.66 1112.66 5 941.03997.55 1030.581111.50 1242.51 1324.641030.581399.361111.501481.781527.621585.091242.51 1324.641399.36 1481.781527.621585.09 902.80 1037.79 922.95 1020.931055.17 922.95 1020.931055.17 5 1112.57 1584.88 5 1584.88 903.74 903.74 1499.92 1499.92 1112.66 1229.89 1271.581325.651399.59 1271.581325.651399.59 1585.09 0 0 1123.931195.561275.72 1325.28 1410.221456.741123.931500.791195.561575.751275.72 1325.28 1410.221456.741500.79 1575.75 1286.761356.781389.82 1513.35 903.74 947.77991.74 1080.381118.591179.171236.271277.93947.77991.74 1351.971425.951080.381118.591179.175 1573.861236.271277.93 1351.971425.95 1573.86 1271.58 1399.59 1466.781513.791590.89 1466.781513.791590.89 0 0 1030.581111.50 1242.51 1324.641399.36 1481.781527.62 922.95 1020.931055.171123.931195.561275.72 1325.28 1410.221456.741500.79 1575.75 0 0 1584.88 0 0 1499.92 1325.65 1466.781513.791590.89 0 0 947.77991.74 1080.381118.591179.171236.271277.931351.971425.95 1573.86 0 0 Octyl 0 400 500 600 700 400800 500900 6001000 7001100 8001200 9001300 10001400 11001500 12001600 1300 1400400 1500500 1600600 700 400800 500900 6001000 7001100 8001200 9001300 10001400 11001500 12001600 1300 14000 1500 1600 400 500 600 700400 800 500 900 600 1000 700 1100 800 1200 900 1300 1000 1400 1100 15000 1200 1600 1300 1400 1500 1600 400 500 600 700400 800500 900600 1000700 1100800 1200900 13001000 14001100 15001200 1600 1300 1400 1500 1600 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 400 500 600 700 800 900 1000 1100m/z 1200 1300 1400 1500m/z 1600 400 500 600 700400 800 500 900 600 1000 700 1100 800 1200 900 1300 1000 1400 1100 1500 1200 1600 1300 1400 1500 1600 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 m/z m/z 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 m/z m/z m/z m/z m/z m/z m/z m/z 5 99 90 100 1.5 >99 100 m/z m/z m/z glucoside No detergent,No detergent, unprocessedNo unprocessed detergent, unprocessed + 1% lauryl+ 1% maltoside,lauryl maltoside,+ 1% unprocessed lauryl unprocessed maltoside, unprocessedLauryl Laurylmaltoside, maltoside, processedLauryl processed maltoside, processed + 1% NP-40,+ 1% NP-40, unprocessed+ unprocessed 1% NP-40, unprocessed NP-40,NP-40, processed processedNP-40, processed

Octyl 200 3.0 >99 100 5 99 95 thioglucoside Triton 25 0.375 >95 82 Base peak LC-MS Figure 2. Results using standard Pierce Detergent Removal Spin Column, 0.5 mL. Tryptic digests (0.1 mL, chromatograms Lauryl X-100 100 µg) containing detergent were each processed through 0.5 mL of Pierce Detergent Removal Resin and subjected 1 98 99 maltoside (1%) 50 0.75 >95 86 to LC-MS/MS analysis. Top row: Base peak LC-MS chromatograms. Bottom row: Integrated mass spectra. Similar ™ 100 1.5 >95 86 Integrated results were produced for Brij -35 detergent, octyl glucoside, octyl thioglucoside, and SDS (data not shown). Triton X-114 2 95 100 mass spectra 200 3.0 >95 93 Brij-35 1 99 97 NP-40 25 0.375 95 90 Tween 20 0.25 99 87 (0.75%) 50 0.75 96 94 Peptide identification results

96-well SDS 5 99 89 100 1.5 97 91 Spin 80 Number of unique peptides Plate Triton X-100 4 99 100 200 3.0 97 97 Sequence coverage (%) 900 70 Spectral count CHAPS 25 0.375 95 64 800 NP-40 1 95 100 Peptide count (1%) 60 50 0.75 97 70 700 CHAPS 5 99 100 50 100 1.5 98 78 600 500 200 3.0 98 75 40 400 30

Number of peptides of Number 300 LC/MS analysis of digested BSA sample 20 200

10 100

0 SDS-processed SDS-unprocessed No SDS-unprocessed

No detergent- No detergent-

unprocessed (2.5 µg) SDS-processed (2.5 µg) unprocessed (10 µg) SDS-processed (10 µg) SDS-unprocessed (2.5 µg) SDS-unprocessed (10 µg)

No detergent-processed (2.5 µg) No detergent-processed (10 µg)

Figure 3. Results using HiPPR Detergent Removal Resin. BSA (25 Figure 4. Results using Pierce Detergent Removal Spin Columns, and 100 μg/mL) was digested in the presence and absence of detergents 0.5 mL. A tryptic digest of HeLa cell lysate (0.1 mL, 100 μg) containing and the samples were processed for LC-MS/MS analysis. Effective 1% SDS was processed through 0.5 mL of Pierce Detergent Removal detergent removal resulted in greater peptide identification and high Resin and subjected to LC-MS/MS analysis. The processed sample MASCOT™ scores. allowed similar numbers of identified peptides as digests containing no Figure 1. Results using HiPPR Detergent Removal Resin. BSA (100 μg/mL) tryptic digests were prepared without detergent, in the presence of 0.5% SDS. Peptide identification is greatly reduced in sample containing SDS. Triton X-100 or spiked with 0.5% SDS following enzymatic digestion. Samples (0.1 mL) containing detergent were processed with the HiPPR Detergent Effective detergent removal enables greater peptide identification. Removal Resin and compared to unprocessed or detergent-free samples by LC-MS/MS. Results demonstrate that detergent removal is effective and produces results similar to those observed for samples containing no detergent.

26 27 Detergent removal using chromatography resins Protein clean-up technical handbook

Selecting the right format for sample processing Spin columns, loose resin and kits

The Thermo Scientific Pierce Detergent Removal Resins Highlights: The HiPPR Detergent Removal Resin is available in a sample processing; simply remove storage buffer, wash are provided in convenient spin column or plate formats • High performance—removes detergent with >90% pre-dispensed 0.1 mL format or as a kit with bulk resin and resin with equilibration buffer, add sample, incubate, and that quickly and efficiently remove ionic, nonionic and/or recovery and no sample dilution empty spin columns for customizing filling and processing. obtain detergent free sample upon final centrifugation. The zwitterionic detergents from protein or peptide samples The Pierce Detergent Removal Resin is available in four resin is also available in a loose resin (10 mL pack size) for • Versatile—effectively removes a wide variety of to improve compatibility with downstream applications. convenient pre-packed column sizes for quick and easy customized applications or columns. detergents from peptide or protein samples Two formulations are available that are optimized to remove detergents from peptide samples with different • Optimized—separate formulations for samples with concentration ranges. The HiPPR (High Protein and Peptide peptide concentrations 100 µg/mL Recovery) products are recommended for peptide samples • Flexible—available in various formats, including spin 100 µg/mL.

• Convenient—simple method that improves MS Discard Discard peptide coverage 1. Centrifuge for 1 minute 2. Add 0.4 mL equilibration buffer, 3. Add detergent-containing 4. Centrifuge at 1,500 x g for 2 minutes at 1,500 x g to remove centrifuge at 1,500 x g for 1 minute sample (25–100 µL) and to collect the detergent-free sample the storage buffer. and discard the flow-through. incubate for 2 minutes at RT. for downstream applications. Table 3. Detergent removal product selection guide Repeat two additional times.

HiPPR HiPPR HiPPR Detergent Protocol summary for Thermo Scientific Pierce Detergent Removal Spin Columns (0.5 mL). Detergent Detergent Detergent Removal Spin Detergent Removal Spin Removal Spin Removal Spin Columns Detergent Removal Spin Columns Kit Plates (4 sizes) Removal Resin Plates

96-well spin plates

Sample 100 µL 25–200 µL 100 µL 10–25 µL 10–2,500 µL 20–100 μL The pre-packed Thermo Scientific™ HiPPR™ and Pierce™ peptide recovery as the spin column format. Each plate volume 25–100 µL range(s) 150–500 µL 96-well Detergent Removal Spin Plates do not require resin can process up to 96 samples simultaneously, using 25 to 500–1,000 µL hydration or dispensing, and offer the same high protein and 100 µL of sample per well. Recommended 1–100 µg/mL 1–100 µg/mL 1–100 µg/mL >100 µg >100 µg >100 µg peptide sample concentration range

Format Pre-filled spin 5 mL resin + 96-well pre-filled Pre-filled spin 10 mL resin 96-well pre-filled columns 0.8 mL empty spin plate columns spin plate spin columns

Resin bed 0.1 mL 0.025–0.2 mL 0.1 mL 0.125 mL 0.125 mL–10 mL 0.55 mL volume(s) 0.5 mL 1. Remove the bottom seal and stack 2. Add 300 µL of buffer to each well and 3. Stack the detergent-removal plate on top 4. Recover the detergent-free sample 2 mL the detergent-removal plate on top centrifuge. Discard the flow-through. of a sample-collection plate. Apply sample for downstream analysis. of a wash plate. Remove the top seal Repeat this step two times. and incubate at room temperature for 4 mL and centrifuge.* 2 minutes. Centrifuge to remove detergent.

* Centrifugations are performed for 2 minutes at 1,000 x g.

Protocol summary for Thermo Scientific Pierce Detergent Removal Spin Plates.

Learn more at thermofisher.com/detergentremoval

28 29 Protein concentration using ultrafiltration membranes Protein clean-up technical handbook

Protein concentration using Selecting the right membrane low fouling characteristics, exceptional flux and broad pH range. Regenerated cellulose membranes are highly ultrafiltration membranes hydrophillic and may show higher protein recovery than other membranes when processing very dilute solutions. Regenerated cellulose is resistant to autoclaving, can Dilute protein solutions can be concentrated using chemicals be cleaned and re-used, and has extended chemical Overview such as ammonium sulfate, trichloroacetic acid (TCA) or resistance. Cellulose triacetate (CTA) membranes have potassium chloride/sodium dodecyl sulfate, or through high hydrophilicity and very low nonspecific binding that Traditional dialysis or gel filtration freeze-drying (lyophilization). However, chemical protein characterizes this membrane. Cast without any membrane precipitation may reduce protein activity and yield. In support that could trap or bind passing micro solutes, these is effective for contaminant addition, these agents are contaminants, often requiring membranes are preferred for sample cleaning and protein removal for downstream applications. Although lyophilization removal and when high recovery of the filtrate solution is of removal and buffer exchange, but does not create these issues, it is more time consuming than primary importance. other methods. additional sample processing is Devices for fast concentration and high The preferred method for the rapid concentration and protein recovery often required to concentrate dilute buffer exchange (diafiltration) of small to mid-volume protein Thermo Scientific Pierce Protein Concentrators were samples is using centrifugal concentrators containing designed to provide rapid sample processing and high protein samples. Following dialysis, ultrafiltration membranes. During concentration, both liquid recovery over a wide range of sample volumes the regenerated cellulose tubing (buffers) and low molecular weight solutes are forced The most commonly used membranes for protein (100 µL–100 mL). These easy-to-use devices contain a high through the membrane where they are collected on the concentration devices are polyethersulfone (PES), performance PES membrane and are available in multiple or device can be submerged into other side (filtrate). Macromolecules remain on the sample regenerated cellulose, and cellulose triacetate (CTA). sizes to process different sample volumes efficiently and side of the membrane, where they become concentrated to PES has a uniform surface devoid of hydrophobic or effectively. A range of molecular weight cutoffs–3K, 5K, a hygroscopic reagent (such as a smaller volume (retentate) as the reagent is forced across hydrophillic interactions and offers excellent protein 10K, 30K, and 100K MWCOs are available to accommodate the membrane to the opposite side. For buffer exchange, recovery for most solutions. PES membranes exhibit proteins as small as 6K or greater than 600K MWCO. polyethylene glycol) to draw the the retentate is diluted to the original volume with exchange Table 1. Comparison of protein recovery for the Thermo Scientific Pierce Protein Concentrators based on molecular weight cutoff buffer and centrifuged. This can be repeated until the (3K, 10K, 30K, and 100K MWCO) solution across the membrane desired level of exchange or desalting has been achieved. Protein 3K MWCO 10K MWCO 30K MWCO 100K MWCO and concentrate the sample. This Percent Recovery (%) combined dialysis and dehydration Aprotinin, 6.5 kDa (0.25 mg/mL) 97 5 Ubiquitin, 8.5 kDa (0.1 mg/mL) 93 method is gentle but time intensive. Cytochrome C, 12 kDa, (0.25 mg/mL) 95 92 6.5 The preferred method for the rapid Carbonic anhydrase, 30 kDa (0.25 mg/mL) 95 95 Ovalbumin, 45 kDa, (0.25 mg/mL) 94 96 concentration and buffer exchange BSA, 66 kDa, (0.25 mg/mL) 98 98 (0.1 mg/mL) = 96 23 (0.25 mg/mL) = 98

(diafiltration) of small to mid- IgG, 150 kDa, (0.25 mg/mL) 94 98 volume protein samples is using Fibrinogen, 340 kDa, (0.25 mg/mL) 92 Figure 1. Overview of sample processing steps using Ferritin, 440 kDa, (0.1 mg/mL) 97 Thermo Scientific™ Pierce™ Protein Concentrators. centrifugal concentrators containing Apoferritin, 480 kDa, (0.4 mg/mL) 85

Thyroglobulin, 660 kDa (0.05 mg/mL) (0.05 mg/mL) = 98 ultrafiltration membranes. (0.25 mg/mL) = 98

30 31 Protein concentration using ultrafiltration membranes Protein clean-up technical handbook

Membrane performance data Flux rates for different MWCOs and sizes 0.5 mL concentrators 6 mL concentrators Protein recovery compared to other vendors 120 120 3K flux rate 3K flux rate 10K flux rate 10K flux rate 0.5 mL concentrators 6 mL concentrators 100 100 30K flux rate 30K flux rate 100 100 98 98 100 97 100K flux rate 100K flux rate 90 94 95 90 80 80 90 91 92 90 93 91 90 80 85 80 86 60 60 70 70 60 60 59 40 40 50 50 % Volume decrease Volume % 40 40 decrease Volume % 20 20 30 30 27 0 0 20 20 0 2.5 5 7.5 10 12.5 15 0 15 30 45 60 75 90 Protein recovery (%) recovery Protein Protein recovery (%) recovery Protein 10 10 Spin time (minutes) Spin time (minutes) 0 0 3K MWCO 10K MWCO 30K MWCO 3K MWCO 10K MWCO 30K MWCO 100K MWCO Aprotinin (6.5 kDa) Protein (46 kDa) Protein A (46 KDa) Aprotinin Carbonic anhydrase Ovalbumin Fibrinogen 20 mL concentrators 100 mL concentrators (6.5 kDa) (30 kDa) (45 KDa) (340 kDa) Thermo Scienti c 0.5 mL Millipore 0.5 mL Pall 0.5 mL 120 120 Thermo Scienti c 6mL Millipore 4 mL 3K flux rate 3K flux rate 10K flux rate 10K flux rate 100 100 20 mL concentrators 100 mL concentrators 30K flux rate 30K flux rate 100K flux rate 100K flux rate 100 100 80 80 97 98 97 98 90 96 92 90 95 93 90 92 91 60 60 86 89 80 85 80 81 70 70 76 40 40 60 60 % Volume decrease Volume % 50 50 decrease Volume % 20 20 40 40 0 0 30 30 0 15 30 45 60 75 90 0 5 10 15 30 45 60 90 20 20 Spin time (minutes) Spin time (minutes) Protein recovery (%) recovery Protein Protein recovery (%) recovery Protein 10 10 0 0 Figure 4. Comparison of flux rates for different MWCOs using 0.5 mL concentrators: 3 minutes for 10K, 30K, and 100K and 15 minutes 3K MWCO 10K MWCO 30K MWCO 100K MWCO 3K MWCO 10K MWCO 30K MWCO 100K MWCO Aprotinin Carbonic anhydrase Ovalbumin Fibrinogen Aprotinin Carbonic anhydrase Ovalbumin Fibrinogen 0.5 mL, 6 mL, 20 mL, and 100 mL concentrators. Samples of protein for 3K. 6 mL concentrators: 15 minutes for 10K, 30K, and 100K, and 90 (6.5 kDa) (30 kDa) (45 KDa) (340 kDa) (6.5 kDa) (30 kDa) (45 KDa) (340 kDa) for each MWCO (Cytochrome C, 12 kDa (3K); Ovalbumin, 45 kDa (10K); minutes for 3K. 20 mL concentrators: 15 minutes for 10K, 30K, and 100K, Thermo Scienti c 20 mL Millipore 15 mL Thermo Scienti c 20 mL Millipore 15 mL BSA, 66 kDa (30K); and Thyroglobulin, 660 kDa (100K), at approximately and 60 minutes for 3K. 100 mL concentrators: 15 minutes for 10K, 30K, Figure 2. Comparison of protein recovery between Pierce instructions: 0.5 mL (15,000 x g), 6 mL (4,000 x g), 20 mL (4,700 x g), and 0.25 mg/mL starting concentrations) were centrifuged in Pierce Protein and 100K, and 90 minutes for 3K. Concentrators (using 3K, 5K, 10K, 30K, or 100K MWCO) and other 100 mL (1,200 x g) until a greater than 15- to 30-fold decrease in sample Concentrators to determine the rate at which >90% protein is concentrated. vendors for 0.5 mL, 6 mL, 20 mL, and 100 mL concentrators. volume was achieved. Protein concentration was measured by either Samples of different protein solutions were centrifuged in Pierce Pierce BCA Protein Assay (0.5 mL concentrators only) or absorbance

Protein Concentrators and other suppliers’ concentrators according to at A280. Chemical compatibility

The polyethersulfone membranes used in the Pierce Protein Concentrators and salts (Table 2). Samples containing high levels of cell membranes, fats, are compatible with most standard aqueous biological samples, buffers, or lipids may reduce performance and result in membrane blockage. A. B. AEBSF Protease/excipient mixture AEBSF Flow-through Table 2. Concentrator chemical compatibility* 14.707 14.789 500 500 Acids & Bases Rating Organics Rating Miscellaneous Rating

17.817 bestatin bestatin Acetic acid (25%) A Acetone NR Ammonium sulfate (saturated) A 400 400 17.812 Formic acid (5%) A Acetonitrile NR Glycerine (70%) A Hydrochloric acid (1 M) A Benzene (100%) NR Guanidine HCL (6 M) A 300 300 Lactic acid (5%) A Chloroform (1%) NR Imidazole (300 mM) A mAU leupeptin mAU leupeptin Nitric acid (10%) A Dimethyl sulfoxide (5%) A Phosphate buffer (1 M) A excipient 200 200 excipient Sodium hydroxide (2.5 M) NR Ethanol (70%) A Polyethylene glycol (10%) A 18.872 Sulfamic acid (5%) A Ethyl acetate (100%) NR Sodium carbonate (20%) A

17.070 Trifluoroacetic acid (10%) A Formaldehyde (30%) A Sodium deoxycholate (5%) A 100 100 17.051 22.442 19.327 Hydrocarbons (aromatic) NR Sodium dodecylsulfate (0.1 M) A 16.002 16.642 18.404 0 0 Hydrocarbons (chlorinated) NR Sodium hypochlorite (200 ppm) A 1 15 20 1 15 20 Isopropanol (70%) A Sodium nitrate (1%) A Time (minutes) Time (minutes) Mercaptoethanol (1 M) NR Tween 20 (0.1%) A Pyridine (100%) NR Triton X-100 (0.1%) A Figure 3. Separation of polymers from small molecules using Pierce were centrifuged for 10 minutes at 13,000 x g. The flow-through was Protein Concentrators PES, 100K MWCO, 0.5 mL. The excipient, collected and compared to the non-concentrated controls using reverse- Tetrahydrofuran (5%) NR Urea (8 M) A sodium starch glycolate (MW >500,000 to 11,000,000), was separated phase HPLC. HPLC conditions: column = Synergi 4u Hydro-RP 80A, Toluene (1%) NR from a mixture of protease inhibitors leupeptin (MW 475.6), AEBSF Gradient Buffer A = 0.1% TFA in water; Gradient Buffer B = 0.1% TFA in A = Acceptable NR = Not Recommended (MW 239.69), and bestatin (MW 345) using the Pierce Protein Concentrator acetonitrile. Panel A. HPLC chromatogram of the excipient and protease *Concentrations listed are provided as guidelines and do not necessarily represent maximum tolerances. Some compatible chemicals might modify the apparent molecular weight of molecules in the sample PES, 100K MWCO, 0.5 mL. The excipient and the inhibitors with excipient inhibitor mixture. Panel B. HPLC chromatogram of the mixture after and/or the molecular weight cutoff rating of the membrane. were dissolved in 0.15 M NaCl. Half of the volume was loaded into the removal of the excipient using the concentrator. Peaks are identified concentrator after pre-wetting the membrane with 0.15 M NaCl. Samples and labeled. 32 33 Protein concentration using ultrafiltration membranes Protein clean-up technical handbook

Selecting the right format Highlights: Pierce Protein Concentrators, 0.5 mL The Thermo Scientific Pierce Protein Concentrators, • Rapid processing—unique design minimizes membrane 0.5 mL, are ideal for processing samples between fouling and sample concentration of 10- to 30-fold can 100 and 500 μL. The 0.5 mL concentrators are available be achieved in 5–30 minutes for 10K MWCO (device in 3K, 10K, 30K, and 100K MWCO. These devices are dependent—times may vary for other MWCOs), even with compatible with most benchtop microcentrifuges with fixed- particle laden solutions. angle rotors that accommodate 2.2 mL tubes. Centrifuge at 15,000 x g until the desired concentration factor is achieved. • High recovery—retain >90% of protein samples while Up to a 30-fold concentration can be obtained in as little as removing contaminants or exchanging buffers. 10 minutes with protein solutions of 0.1 mg/mL or higher, but • Convenient—clear markings, wide sample chamber, and times may vary significantly based on molecular weight cutoff removable filtrate chamber make handling simple and easy. and sample concentration or viscosity. The dead-stop volume is approximately 15 μL. Typical protein recovery is >90%. • Instrument compatible—can be used with standard centrifuges utilizing fixed-angle or swinging-bucket rotors. Table 4. Processing time and protein recovery using Thermo Scientific Pierce Protein Concentrators, 0.5 mL. Four different protein solutions The Pierce concentrators are designed for easy handling starting with a 0.5 mL volume were concentrated over 30-fold in a fixed angle rotor at 15,000 x g at 25°C, using 3K, 10K, 30K, or 100K MWCO devices. The Thermo Scientific Pierce Protein Concentrators are and sample processing. The upper chamber is wide enough Membrane Processing Protein retentate Protein retentate MWCO (kDa) Protein solute time (minutes) volume (µL) recovery (%) easy-to-use centrifugal devices that enable fast processing for convenient sample addition. Following concentration, and excellent recovery of protein samples. These disposable the sample chamber can be simply detached from the 3 Cytochrome C, 12 kDa (0.25 mg/mL) 15 67 100 ultrafiltration devices contain a polyethersulfone (PES) filtrate chamber, and the concentrated protein can be easily 10 Ovalbumin, 45 kDa (0.25 mg/mL) 3 10 95 membrane for the concentration, desalting, and buffer removed with a pipette tip. The screw top cap eliminates 30 BSA, 66 kDa (0.25 mg/mL) 3 48 96 exchange of biological samples, such as tissue culture the need to use parafilm when mixing solutions during 100 Thyroglobulin, 660 kDa (0.25 mg/mL) 11 9 100 media, antiserum, monoclonal antibody preparations, and buffer exchange. In addition, unlike similar products from chromatography fractions. They can also be used to remove other vendors, reverse centrifugation is not required to unincorporated label following protein modification reactions. recover concentrated sample from the device. Selecting The PES membrane in these devices is available in five the appropriate MWCO will depend on the size of your Pierce Protein Concentrators, 6 mL The Thermo Scientific Pierce Protein Concentrators, distinct molecular weight cutoffs (MWCO) of 3K, 5K, 10K, protein. The PES membrane has been rated for retaining 6 mL, are ideal for processing samples between 2 mL and 30K, and 100K, and can be used for processing sample molecules with a molecular weight at least two-fold greater 6 mL. These 6 mL concentrators are available in 3K, 10K, volumes from 100 μL to 100 mL. The MWCOs are etched on than the MWCO rating of the membrane within the device. 30K, and 100K MWCO. These devices fit into a swinging the sides of the concentrators for easy identification, while Reduced recovery may occur when using a concentrator bucket or a fixed angle rotor that accommodates 15 mL a clear window with graduations on the side of each device with molecules smaller than the recommended MWCO. conical tubes. Centrifuge at 3,000 to 4,000 x g until the allows for visual determination of the concentrated sample Protein recovery will vary depending on the specific protein desired concentration factor is achieved. Greater than (retentate) volume. The unique design provides reliable and in the sample and its starting concentration. To achieve 30-fold concentration can be obtained in as little as consistent results. Multiple device sizes are available to >90% recovery of protein, the minimum protein sample 15 minutes with protein solutions of 0.1 mg/mL or higher; handle maximum sample volumes of 0.5 mL, 6 mL, 20 mL, concentration should be 0.05 mg/mL. however, times may vary significantly based on molecular and 100 mL. weight cutoff and sample concentration or viscosity. The Table 3. Thermo Scientific Pierce Protein Concentrators selection guide. dead-stop volume is approximately 30 μL. Typical protein recovery is >90%. Volume Range 0.1 mL– 0.5 mL 2 mL–6 mL 5 mL–20 mL 20 mL–100 mL

Table 5. Processing time and protein recovery using Thermo Scientific Pierce Protein Concentrators, 6 mL. Four different protein solutions starting with a 6 mL volume were concentrated over 30-fold in a swinging bucket rotor at 4,000 x g at 25°C, using 3K, 10K, 30K, or 100K MWCO devices. Membrane Processing time Protein retentate Protein retentate MWCO (kDa) Protein solute (minutes) volume (µL) recovery (%) MWCOs available 3K, 10K, 30K, 100K 3K, 10K, 30K, 100K 3K, 10K, 30K, 100K 5K, 10K, 30K, 100K 3 Cytochrome C, 12 kDa (0.25 mg/mL) 90 174 94 Processing time* 3–15 min 15–90 min 15–60 min 15–90 min 10 Ovalbumin, 45 kDa (0.25 mg/mL) 15 91 98 Retentate volume range* 9–67 µL 51–174 µL 121–777 µL 1.9–3.5 mL 30 BSA, 66 kDa (0.25 mg/mL) 15 51 100 Protein recovery range* 95–100% 94–100% 94–100% 92–98% 100 Thyroglobulin, 660 kDa (0.25 mg/mL) 15 213 97 * Four different proteins solutions were used for each molecular weight cutoff (MWCO). 34 35 Protein concentration using ultrafiltration membranes Endotoxin removal using affinity chromatography Protein clean-up technical handbook

Pierce Protein Concentrators, 20 mL The Thermo Scientific Pierce Protein Concentrators, 20 mL, are ideal for processing samples between 5 mL Endotoxin removal using affinity and 20 mL. These 20 mL concentrators are available in 3K, 10K, 30K, and 100K MWCO. These devices fit into a swinging bucket or a fixed angle rotor that accommodates chromatography 50 mL conical tubes. Centrifuge at 3,000 to 5,000 x g until the desired concentration factor is achieved. Up to a 30-fold concentration can be obtained in as little as 15 minutes with protein solutions of 0.1 mg/mL or higher; however, times Overview may vary significantly based on molecular weight cutoff and sample concentration or viscosity. The dead-stop volume is Biotechnology refers to biological approximately 50 μL. Typical protein recovery is >90%.

Table 6. Processing time and protein recovery using Thermo Scientific Pierce Protein Concentrators, 20 mL. Four different protein solutions processes that have been starting with a 20 mL volume were concentrated over 30-fold in a swinging bucket rotor at 4,700 x g at 25°C, using 3K, 10K, 30K, or 100K MWCO devices. Membrane Processing time Protein retentate Protein retentate engineered. Following the MWCO (kDa) Protein solute (minutes) volume (µL) recovery (%) 3 Cytochrome C, 12 kDa (0.25 mg/mL) 60 121 100 development of recombinant DNA 10 Ovalbumin, 45 kDa (0.25 mg/mL) 15 528 94 30 BSA, 66 kDa (0.25 mg/mL) 15 216 100 technology, peptides, hormones, 100 Thyroglobulin, 660 kDa (0.25 mg/mL) 15 777 98 and proteins that were originally Endotoxin contamination is a common problem with extracted from tissues and recombinant proteins purified from gram-negative bacteria Pierce Protein Concentrators, The Thermo Scientific Pierce Protein Concentrators, such as E. coli. Endotoxins are heat-stable molecules 100 mL, are ideal for processing samples between 20 mL secretions can now be produced associated with the outer membranes of certain gram- 100 mL and 100 mL. These 100 mL concentrators are available in negative bacteria. When these bacterial cells die, their cell 5K, 10K, 30K, and 100K MWCO. The devices can be used synthetically with high purity and membranes rupture and endotoxins (which are essential directly for volumes under 90 mL; when adding volumes lipopolysacchride (LPS) components of the cell walls) are between 90 mL and 100 mL, it is recommended to parafilm yield. Protein-based engineering released into the surrounding environment. Endotoxins are the cap to the bottle to help prevent leakage. These frequent contaminants of protein solutions derived from devices fit into a swinging bucket or a fixed angle rotor that is now one of the fastest growing bioproduction. Endotoxins are also toxic to cells grown in accommodates 250 mL bottles. Centrifuge at 1,200 x g until tissue culture. Traditional endotoxin removal methods and the desired concentration factor is achieved. Up to a 30-fold areas in research. their limitations are described in Table 1. concentration can be obtained in as little as 15 minutes with protein solutions of 0.1 mg/mL or higher; however, times Table 1. Benefits of Thermo Scientific™ Pierce™ High Capacity Endotoxin Removal Resin over traditional methods. may vary significantly based on molecular weight cutoff and sample concentration or viscosity. The dead-stop volume is Traditional endotoxin Benefit from Pierce High Capacity removal method Limitations Endotoxin Removal Resin approximately 350 μL. Typical protein recovery is >90%. Anion-exchange Successfully process proteins across a range Loss of negatively charged protein chromatography of isoelectric points Table 7. Processing time and protein recovery using Thermo Scientific Pierce Protein Concentrators, 100 mL. Four different protein solutions starting with a 90 mL volume were concentrated over 30-fold in a swinging bucket rotor at 1,200 x g at 25°C, using 5K, 10K, 30K, or 100K MWCO devices. Only removes large endotoxin aggregates, so it is compatible only with low molecular weight proteins. Endotoxin bound to Successfully process proteins ranging in Membrane Processing time Protein retentate Protein retentate Ultrafiltration MWCO (kDa) Protein solute (minutes) volume (mL) recovery (%) toxin will not be effectively removed. Technique also exerts molecular weight from 12 to 150 kDa strong physical forces on the protein 3 Cytochrome C, 12 kDa (0.25 mg/mL) 90 1.9 98 Resin binds up to 2,000,000 EU/mL† Membrane-based Reduced endotoxin binding capacity compared to resin 10 Ovalbumin, 45 kDa (0.25 mg/mL) 15 3.5 96 and can be reused up to 10 times chromatography based methods; non-reusable with no loss in performance 30 BSA, 66 kDa (0.25 mg/mL) 15 2.8 95

100 Thyroglobulin, 660 kDa (0.25 mg/mL) 15 2.5 92 Polymyxin B Ligand exhibits neurotoxicity and sodium deoxycholate Poly(ε-lysine) is a safe, nontoxic polymer of affinity ligand buffers cause renal tubular necrosis the natural amino acid lysine and is commonly used as a food preservative Learn more at thermofisher.com/concentrators † One endotoxin unit/mL (EU/mL) equals approximately 0.1 ng endotoxin/mL of solution. 36 37 Endotoxin removal using affinity chromatography Protein clean-up technical handbook

Products designed for efficient endotoxin removal Tools to quantitate endotoxin removal Protein compatibility Ultrafiltration, Polymixin B affinity resin, or resin- or It is critical to measure endotoxin levels in protein solutions. Table 3. Clean-up proteins of various molecular weights and isoelectric points with minimal sample. Pierce High Capacity Endotoxin Removal Spin Columns, 0.5 mL, were challenged for 1 hour with different proteins of various molecular weight and charge. In all samples endotoxin removal was membrane-based chromatography are the traditional In the early 1950s Frederick Bang discovered that the >99% and protein recovery was >85%. methods for endotoxin removal. All have limitations in protein horseshoe crab’s blood cells (amoebocytes) contain a Molecular Isoelectric Initial endotoxin Final endotoxin Endotoxin recovery, endotoxin binding capacity, and/or have toxicity clotting agent that attaches to dangerous endotoxins Protein weight point concentration concentration removal (1 mg/mL) (Da) (pI) (EU/mL) (EU/mL) efficiency Protein recovery concerns. Thermo Fisher Scientific offers a modified produced by gram negative bacteria. The Limulus ε-poly-L-lysine [poly(ε-lysine)] affinity resin, which is a safe, Amebocyte Lysate (LAL) endotoxin test gel-clot method Cytochrome C 12,000 10.6 10,000 1.35 >99.9% 86% nontoxic polymer of the natural amino acid lysine that is was developed to measure endotoxin levels (Figure 2), Myoglobin 17,000 6.8 10,000 3.67 >99.9% 87% commonly used as a food preservative (Figure 1). This and was approved by the United States Food and Drug Bovine serum 66,000 4.9 10,000 0.80 >99.9% 85% resin is available as a slurry to pack a custom column, or in Administration (FDA) in 1983. This method can be used to albumin (BSA) convenient pre-packed, single-use spin columns optimized determine if products or materials are “endotoxin free”. Bovine gamma 150,000 7.4 10,000 4.60 >99.9% 92% for different sample volumes. globulin (BGG) As biotechnology has evolved, methods have been Cationic developed to successfully reduce the contamination of Hydrophobic Endotoxin removal compared to other suppliers NH3 unwanted pathogens and increase the production volumes Table 4. Thermo Scientific Pierce High Capacity Endotoxin Removal Resin delivers the best sample clean-up. Various endotoxin removal OH resins were challenged with 2 mL of 1 mg/mL BSA containing 25,000 EU/mL according to the respective manufacturers’ procedures. The poly(ε-lysine)- O N of these endotoxin-free proteins. bead based Pierce High Capacity Endotoxin Removal Resin consistently exhibited the highest endotoxin removal efficiency and the highest protein recovery.

Cellulose n = 30-40 OH H O Initial endotoxin Final endotoxin Protein concentration (EU/mL) concentration (EU/mL) recovery Poly(ε-lysine) resin Factor C End (from LAL) o to High Capacity Endotoxin Removal Resin x Thermo Scientific Pierce High Capacity in 25,000 <1 91% Ac-Ile-Glu-Ala-Arg-pNA Endotoxin Removal Resin Figure 1. The poly(ε-lysine) affinity ligand binds endotoxins through (Synthetic peptide chromogenic substrate) Thermo Scientific Detoxi-Gel both ionic and hydrophobic interactions. The multiple ε-aminobutyl Active 25,000 77 74% Endotoxin Removal Gel [Thermo Fisher Scientific] groups impart both a positive charge via the primary amines as well as a Protease hydrophobic characteristic via the butyl spacer between primary amines. Affi-Prep Polymyxin Matrix [Bio-Rad] 25,000 13 88% The hydrophilic nature of the porous cellulose base–matrix is masked Standard curve by thorough derivatization of its interior and exterior surfaces with the Polymyxin B-Agarose [Sigma] 25,000 900 63% 405 poly(ε-lysine) ligand. A pNA Absorbance ReductEtox [Sterogene] 25,000 13,900 92% at 405 nm + EndoTrap red [Hyglos] 25,000 64 82%

Ac-Ile-Glu-Ala-Arg EU/mL Sample compatibility Figure 2. LAL endotoxin quantitation assay overview. Table 5. Thermo Scientific Pierce High Capacity Endotoxin Removal Resin removes endotoxins from various strains of E. coli. Pierce High Capacity Endotoxin Removal Spin Columns, 0.5 mL, were challenged with 1 mL BSA (1 mg/mL) spiked with 10,000 EU from different strains of E. coli. In all samples removal was performed with >99% efficiency. Resin performance data Initial endotoxin Final endotoxin Endotoxin removal E. coli strain concentration (EU/mL) concentration (EU/mL) efficiency Protein recovery Binding capacity 011:B4 10,000 0.36 >99.9% 83% Table 2. Efficiently remove endotoxins from heavily contaminated samples. Thermo Scientific™ Pierce™ High Capacity Endotoxin Removal Spin Columns, 0.5 mL, were challenged for 1 hour with protein (2 mL of 1 mg/mL BSA) containing 5,000 to 500,000 EU/mL from E. coli strain 055:B5. Even at 026:B6 10,000 4.18 >99.9% 88% the highest endotoxin levels clean-up was >98%. 0127:B8 10,000 0.87 >99.9% 87% Initial endotoxin Final endotoxin concentration (EU/mL)* concentration (EU/mL) Endotoxin removal efficiency Protein recovery 0128:B12 10,000 5.01 >99.9% 88%

5,000 <1 99.98% >90% Table 6. Process protein and antibody samples from a variety of sources. Protein samples from cell culture supernatants, E. coli lysates or 12,500 <1 99.99% >90% human serum were processed with the Pierce High Capacity Endotoxin Removal Spin Columns. In all samples endotoxin levels were reduced to less than 1 EU/mL. 25,000 1.26 99.99% >90% Initial endotoxin Final endotoxin concentration 50,000 7.1 99.99% >90% Protein source concentration (EU/mL) (EU/mL)

250,000 32 99.98% >90% Anti-Fractalkine from cell culture supernatant 8.26 <1

500,000 9,600 98.08% >90% His-tag GFP from E. coli 9,780 <1

*One endotoxin unit/mL (EU/mL) equals approximately 0.1 ng endotoxin/mL of solution. IgG from human serum 78 <1

38 39 Endotoxin removal using affinity chromatography Protein clean-up technical handbook

Selecting the right format 10,000 Figure 4. Remove endotoxins from protein samples in 1 hour. 8,000 Minutes EU Pierce High Capacity Endotoxin Removal Spin Columns, 0.5 mL, were • Durable—reuse resin up to 10 times 6,000 0 10,000.00 challenged for different time intervals with 1 mL samples containing 5 86.00 Thermo Scientific Pierce High Capacity Endotoxin Removal 4,000 1 mg/mL BSA spiked with 10,000 EU. The final endotoxin concentration 30 1.65 ™ ™ Resin selectively binds and removes endotoxins from • Selective—recover ≥85% of your protein sample 2,000 60 1.54 was determined using the Thermo Scientific Pierce LAL Chromogenic 180 0.35 Endotoxin Quantitation Kit (Cat. No. PI88282). A one-hour incubation protein, peptide and antibody samples using a modified 50 time provided 99% endotoxin removal, and longer incubation periods • High performance—complies with FDA guidelines by 3 ε-poly-L-lysine [poly(ε-lysine)] affinity ligand. Endotoxin levels accomplished even greater endotoxin removal. reducing final EU concentration to <5 EU/mL in biological samples are reduced by up to 99% in as fast as 2 1 hour using our spin column format, and protein recovery • Fast—our spin column format enables endotoxin LPS concentration (EU) LPS is ≥ 85%. Pierce High Capacity Endotoxin Removal Resin depletion typically within 1 hour 1 is available as a slurry to pack a custom column or in • Clean—single-use spin columns avoid cross convenient pre-packed, single-use spin columns optimized 0 contamination of samples for different sample volumes. 0 30 60 90 120 150 180 • Optimized—spin columns are optimized for different Time (minutes) Highlights: sample volumes • High capacity—bind up to 2,000,000 EU/mL to eliminate • Economical—large-volume discounts available Loose resin >99% of endotoxins

Table 7. Thermo Scientific endotoxin removal products selection guide by format. Pierce High Capacity Endotoxin Removal Resin is available as a slurry in 10, 100 or 250 mL pack sizes for custom Format: Spin column Spin column Spin column Loose resin packing of endotoxin removal columns, and can be used with gravity flow systems or automated chromatography systems with flow rates ranging from 10 to 15 mL/hour.

Learn more at thermofisher.com/endotoxinremoval

Sample volume 0.5–1 mL 1–4 mL 2–10 mL Varies processing

Supplied as 25% slurry in 20% ethanol 25% slurry in 20% ethanol 25% slurry in 20% ethanol 50% slurry in 20% ethanol Endotoxin quantitation kit concentration in a sample is measured via a chromogenic

Packaging options 5 or 25 columns/pack 5 or 25 columns/pack 5 or 25 columns/pack 10, 100 or 250 mL bottles signal generated in the presence of endotoxins. Samples can be measured on a microplate absorbance reader at 405 nm. A standard curve is created using the E. coli Spin columns endotoxin standard included with each kit to calculate endotoxin levels as low as 0.1 EU/mL (Figure 4). Protein and The pre-packed spin column format is a fast, single-use for >85% protein recovery. Three pre-packed spin- antibody samples can be assayed in as few as 30 minutes. method to remove 99% of endotoxins from protein samples column sizes are available to process protein samples of in as fast as 1 hour (Figure 3). These spin columns use a different volumes. Highlights: batch format to bind and remove endotoxins while allowing • Sensitive—detect as little as 0.1 EU/mL

• Fast—perform this assay in 30 minutes

• Economical—assay requires only 10 μL of a protein sample The Thermo Scientific Pierce LAL Chromogenic Endotoxin • Accurate—E. coli O111:B4 standard in each kit Quantitation Kit measures the amount of endotoxin in a enables accurate endotoxin quantitation protein, peptide or antibody sample using the LAL assay. Designed for up to 50 samples, this quantitation kit is • Versatile—405 nm absorbance reading is 1. Centrifuge at 500 x g for 2. Regenerate with 0.2 N 3. Wash with 2 M NaCl 4. Equilibrate with ET-free 5. Add sample and incubate 6. Centrifuge at 500 x g 1 minute to remove the NaOH (overnight) or followed by ET-free buffer, pH 6–8. Repeat at 4°–22°C with gentle for 1 minute to collect ideal for processing a few samples at a time compared compatible with common ELISA plate readers storage buffer. 0.2 N NaOH in 95% water. three times. end-over-end mixing for the sample. to other high-throughput quantitation kits. The endotoxin ethanol for 1 to 2 hours. 1 hour. Figure 3. Protocol for Pierce High Capacity Endotoxin Removal Spin Columns.

40 41 Ordering Information Protein clean-up technical handbook

Ordering information Ordering information

Product Quantity Cat. No. Product Quantity Cat. No. Product Quantity Cat. No. Product Quantity Cat. No.

2K MWCO membrane products 3.5K MWCO membrane products (continued) 10K MWCO membrane products (continued) 20K MWCO membrane products Slide-A-Lyzer MINI Dialysis Devices, 10–100 μL 50 devices PI69580 96-well Deep-well Plate, 2.2 mL 1 plate PI88261 Slide-A-Lyzer Dialysis Flask, 250 mL 4 flasks PI87762 Slide-A-Lyzer MINI Dialysis Devices, 0.1–0.5 mL 25 devices PI88402 Sufficient caps are included Formulation: Polypropylene deep-well assay plate SnakeSkin Dialysis Tubing, 22 mm 35 ft./pkg PI68100 Slide-A-Lyzer MINI Dialysis Devices, 2 mL 25 devices PI88405 Sufficient for: 96 microdialysis devices Slide-A-Lyzer MINI Dialysis Devices, 10–100 μL 250 devices PI69553 Sufficient for: Approx. 19 mL per 5 cm length Slide-A-Lyzer G2 Dialysis Cassettes, 30 mL 6 cassettes PI87737 Sufficient caps are included Pierce Plate Seal for Pierce 96-well 1 sheet PI88269 (plus 2.5 cm at each end for closure) yields 100 uses Slide-A-Lyzer G2 Dialysis Cassettes, 70 mL 6 cassettes PI87738 Slide-A-Lyzer G2 Dialysis Cassettes, 30 mL 6 cassettes PI87720 Microdialysis Plates SnakeSkin Dialysis Tubing, 16 mm 35 ft./pkg. PI88243 Formulation: Adhesive coated plastic sheet, Sufficient for: Approx. 10 mL per 5 cm length Slide-A-Lyzer G2 Dialysis Cassettes, 15 mL 8 cassettes PI87736 Slide-A-Lyzer G2 Dialysis Cassettes, 70 mL 6 cassettes PI87721 perforated to 8-well strips fitting microdialysis plate (plus 2.5 cm at each end for closure) yields 100 uses Slide-A-Lyzer G2 Dialysis Cassettes, 0.1–0.5 mL 10 cassettes PI87734 Slide-A-Lyzer G2 Dialysis Cassettes, 15 mL 8 cassettes PI87719 Sufficient for: 12 x 8 microdialysis plate wells SnakeSkin Dialysis Tubing, 35 mm 35 ft./pkg. PI88245 Slide-A-Lyzer G2 Dialysis Cassettes, 0.5–3 mL 10 cassettes PI87735 Slide-A-Lyzer G2 Dialysis Cassettes, 10 cassettes PI87717 7K MWCO membrane products Sufficient for: Approx. 48 mL per 5 cm length 0.25–0.75 mL (plus 2.5 cm at each end for closure) yields 100 uses Slide-A-Lyzer Dialysis Cassettes, 12–30 mL 6 cassettes PI66030 Slide-A-Lyzer G2 Dialysis Cassettes, 1–3 mL 10 cassettes PI87718 Slide-A-Lyzer MINI Dialysis Devices, 10–100 μL 50 devices PI69560 Pierce 96-well Microdialysis Plate, 1-plate set PI88260 Slide-A-Lyzer Dialysis Cassettes, 3–12 mL 8 cassettes PI66012 Sufficient caps are included Slide-A-Lyzer Dialysis Cassettes, 12–30 mL 6 cassettes PI66230 Formulation: Dialysis membrane framed in plastic Slide-A-Lyzer Dialysis Cassettes, 0.1–0.5 mL 10 cassettes PI66005 insert with 96-well deep-well plate Slide-A-Lyzer MINI Dialysis Devices, 10–100 μL 250 devices PI69562 Slide-A-Lyzer Dialysis Cassettes, 0.5–3 mL 10 cassettes PI66003 Slide-A-Lyzer Dialysis Cassettes, 3–12 mL 8 cassettes PI66212 Sufficient caps are included Sufficient for dialysis of 96 samples 10 to 100 μL each Slide-A-Lyzer Dialysis Cassettes, 0.1–0.5 mL 10 cassettes PI66205 Kit contents: 8-microdialysis device strip, 12 strips, Slide-A-Lyzer Dialysis Flasks, 250 mL 4 flasks PI87763 Slide-A-Lyzer G2 Dialysis Cassettes, 0.1–0.5 mL 10 cassettes PI87727 96-well deep-well plate, 1 plate Slide-A-Lyzer Dialysis Cassettes, 0.5–3 mL 10 cassettes PI66203 Slide-A-Lyzer G2 Dialysis Cassettes, 1–3 mL 10 cassettes PI87728 96-well Deep-well Plate, 2.2 mL 1 plate PI88261 Product accessories Slide-A-Lyzer Dialysis Flasks, 250 mL 4 flasks PI87760 Slide-A-Lyzer Dialysis Cassettes, 3–12 mL 8 cassettes PI66710 Formulation: Polypropylene deep-well assay plate Slide-A-Lyzer MINI Dialysis Device Floats 4 floats PI69588 Sufficient for 96 microdialysis devices 3.5K MWCO membrane products Slide-A-Lyzer Dialysis Cassette Kit, 3–12 mL Kit PI66707 Holds 25 MINI Dialysis Units Pierce Plate Seal for Pierce 96-well 1 sheet PI88269 Contains 8 cassettes, 8 buoys and 10 syringes Slide-A-Lyzer Cassette Float Buoys, Grey 8 buoys PI66432 Slide-A-Lyzer MINI Dialysis Devices, 10–100 μL Kit/ PI69558 Slide-A-Lyzer Dialysis Cassettes, 0.1–0.5 mL 10 cassettes PI66373 Microdialysis Plates Holds one 3–12 mL cassette and Float 10 devices Formulation: Adhesive coated plastic sheet, Sufficient caps are included Slide-A-Lyzer Dialysis Cassettes, 0.5–3 mL 10 cassettes PI66370 perforated to 8-well strips fitting microdialysis plate Slide-A-Lyzer Cassette Float Buoys, White 10 buoys PI66430 Sufficient for: 12 x 8 microdialysis plate wells Holds one 0.1–0.5 mL or 0.5–3 mL cassette Slide-A-Lyzer MINI Dialysis Devices, 10–100 μL 50 devices PI69550 Slide-A-Lyzer Dialysis Cassette Kit, 0.5–3 mL Kit PI66372 Sufficient caps are included Contains 10 cassettes, 10 buoys and 10 syringes Slide-A-Lyzer Cassette Carousel Float Buoy 1 buoy PI66431 Gamma-irradiated 10K MWCO membrane Holds ten 0.1–0.5 mL or 0.5–3 mL cassettes Slide-A-Lyzer MINI Dialysis Devices, 10–100 μL 250 devices PI69552 SnakeSkin Dialysis Tubing, 22 mm 35 ft/pkg PI68700 Sufficient caps are included Sufficient for: Approx. 19 mL per 5 cm length Slide-A-Lyzer Dialysis Cassettes, 3–12 mL 8 cassettes PI66453 Slide-A-Lyzer Cassette Syringes, 1 mL* 10 syringes PI66494 (plus 2.5 cm at each end for closure) yields 100 uses Slide-A-Lyzer MINI Dialysis Devices, 0.5 mL 25 devices PI88400 Slide-A-Lyzer Dialysis Cassettes, 12–30 mL 8 cassettes PI66456 Slide-A-Lyzer Cassette Syringes, 5 mL* 10 syringes PI66490 Slide-A-Lyzer MINI Dialysis Devices, 2 mL 25 devices PI88403 10K MWCO membrane products Slide-A-Lyzer Dialysis Cassettes, 0.1–0.5 mL 10 cassettes PI66454 Slide-A-Lyzer Cassette Syringes, 20 mL* 10 syringes PI66493 Slide-A-Lyzer G2 Dialysis Cassettes, 70 mL 6 cassettes PI87726 Slide-A-Lyzer Dialysis Cassettes, 0.5–3 mL 10 cassettes PI66455 Slide-A-Lyzer Dialysis Flotation Disks, for use 6 disks PI87759 Slide-A-Lyzer MINI Dialysis Devices, 10–100 μL 10 devices PI69574 with Slide-A-Lyzer Dialysis Flasks Slide-A-Lyzer G2 Dialysis Cassettes, 30 mL 6 cassettes PI87725 Plus Microtubules Slide-A-Lyzer G2 Dialysis Cassettes, 0.5 mL 10 cassettes PI88250 Reusable; each disk holds 1 flask Sufficient caps are included Slide-A-Lyzer G2 Dialysis Cassettes, 15 mL 8 cassettes PI87724 Slide-A-Lyzer G2 Dialysis Cassettes, 3 mL 10 cassettes PI88251 SnakeSkin Dialysis Tubing Clips 6 clips PI68011 Slide-A-Lyzer MINI Dialysis Devices, 10–100 μL 50 devices PI69570 Slide-A-Lyzer G2 Dialysis Cassettes, 0.1–0.5 mL 10 cassettes PI87722 Slide-A-Lyzer G2 Dialysis Cassettes, 15 mL 8 cassettes PI88252 Sufficient caps are included * Each syringe comes with 18-gauge 1-inch beveled needles. And can also be used with the new Slide-A-Lyzer G2 Dialysis Cassettes, 30 mL 6 cassettes PI88253 Slide-A-Lyzer G2 Dialysis Cassettes. Slide-A-Lyzer G2 Dialysis Cassettes, 1–3 mL 10 cassettes PI87723 Slide-A-Lyzer MINI Dialysis Devices, 10–100 μL 250 devices PI69572 Slide-A-Lyzer Dialysis Cassettes, 0.1–0.5 mL 10 cassettes PI66333 Sufficient caps are included Slide-A-Lyzer G2 Dialysis Cassettes, 70 mL 6 cassettes PI88254 Slide-A-Lyzer Dialysis Cassette Kit, 0.1–0.5 mL Kit PI66335 Slide-A-Lyzer MINI Dialysis Devices, 10–100 μL Kit/ PI69576 Contains 10 cassettes, 10 buoys and 10 syringes Plus Float 10 devices Slide-A-Lyzer Dialysis Cassettes, 0.5–3 mL 10 cassettes PI66330 Sufficient caps are included Learn more at thermofisher.com/dialysis Slide-A-Lyzer Dialysis Cassette Kit, 0.5–3 mL Kit PI66332 Slide-A-Lyzer MINI Dialysis Devices, 0.5 mL 25 devices PI88401 Contains 10 cassettes, 10 buoys and 10 syringes Slide-A-Lyzer MINI Dialysis Devices, 2 mL 25 devices PI88404 Slide-A-Lyzer Dialysis Cassettes, 12–30 mL 6 cassettes PI66130 Slide-A-Lyzer G2 Dialysis Cassettes, 30 mL 6 cassettes PI87732 Slide-A-Lyzer Dialysis Cassettes, 3–12 mL 8 cassettes PI66110 Slide-A-Lyzer G2 Dialysis Cassettes, 70 mL 6 cassettes PI87733 Slide-A-Lyzer Dialysis Cassette Kit, 3–12 mL Kit PI66107 Slide-A-Lyzer G2 Dialysis Cassettes, 15 mL 8 cassettes PI87731 Slide-A-Lyzer Dialysis Flasks, 230 mL 4 flasks PI87761 Slide-A-Lyzer G2 Dialysis Cassettes, 0.1–0.5 mL 10 cassettes PI87729 SnakeSkin Dialysis Tubing, 16 mm 35 ft/pkg PI88242 Slide-A-Lyzer G2 Dialysis Cassettes, 1–3 mL 10 cassettes PI87730 Sufficient for: Approx. 10 mL per 5 cm length plus 2.5 cm at each end for closure) yields 100 uses Slide-A-Lyzer Dialysis Cassettes, 12–30 mL 6 cassettes PI66830 SnakeSkin Dialysis Tubing, 22 mm 35 ft/pkg PI68035 Slide-A-Lyzer Dialysis Cassettes, 0.1–0.5 mL 10 cassettes PI66383 Sufficient for: Approx. 19 mL per 5 cm length Slide-A-Lyzer Dialysis Cassettes, 0.1–0.5 mL 50 cassettes PI66384 (plus 2.5 cm at each end for closure) yields 100 uses Slide-A-Lyzer Dialysis Cassette Kit, 0.1–0.5 mL Kit PI66385 SnakeSkin Dialysis Tubing, 35 mm 35 ft/pkg PI88244 Contains 10 cassettes, 10 buoys and 10 syringes Sufficient for: Approx. 48 mL per 5 cm length Slide-A-Lyzer Dialysis Cassettes, 0.5–3 mL 10 cassettes PI66380 (plus 2.5 cm at each end for closure) yields 100 uses Slide-A-Lyzer Dialysis Cassettes, 0.5–3 mL 50 cassettes PI66381 Pierce 96-well Microdialysis Plate 1-plate set PI88262 Formulation: Dialysis membrane framed in plastic Slide-A-Lyzer Dialysis Cassette Kit, 0.5–3 mL Kit PI66382 insert with 96-well deep-well plate Contains 10 cassettes, 10 buoys and 10 syringes Sufficient for: Dialysis of 96 samples 10 to 100 μL each Slide-A-Lyzer Dialysis Cassettes, 3–12 mL 8 cassettes PI66810 Kit contents: 8-microdialysis device strip, 12 strips, 96-well deep-well plate, 1 plate Slide-A-Lyzer Dialysis Cassettes, 3–12 mL 50 cassettes PI66811 Slide-A-Lyzer Dialysis Cassette Kit, 3–12 mL Kit PI66807 Contains 8 cassettes, 8 buoys and 10 syringes

42 43 Ordering Information Protein clean-up technical handbook

Ordering information Ordering information

Product Quantity Cat. No. Product Quantity Cat. No. Product Quantity Cat. No. Product Quantity Cat. No.

Desalting products Desalting products (continued) Empty spin column products Detergent removal products Zeba Micro Spin Desalting Columns, 25 columns PI89877 Zeba Spin Desalting Columns, 50 columns PI87767 Pierce Micro-Spin Columns 50 columns PI89879 HiPPR Detergent Removal Kit PI88305 7K MWCO, 75 µL 40K MWCO, 0.5 mL Polyethylene filter (30 µm pore size); screw caps Spin Column Kit, 5 mL with O-rings; press-in bottom plugs Sufficient for 25 samples, each 2–12 µL Sufficient for 50 samples, each 70–200 µL HiPPR Detergent Removal 24 columns PI88306 Zeba Spin Desalting Columns, 5 columns PI87768 Sufficient for microcentrifuge procedures with Zeba Micro Spin Desalting Columns, 50 columns PI89878 5–100 µL resin Spin Columns, 0.1 mL 40K MWCO, 2 mL 7K MWCO, 75 µL HiPPR Detergent Removal 2 plates PI88307 Sufficient for 50 samples, each 2–12 µL Sufficient for 5 samples, each 200–900 µL Pierce Centrifuge Columns, 0.8 mL 50 columns PI89868 Polyethylene filter (30 µm pore size); screw-top caps 96-well Spin (filter) Plates, 0.1 mL Zeba Spin Desalting Columns, 25 columns PI89882 Zeba Spin Desalting Columns, 25 columns PI87769 Sufficient for microcentrifuge procedures with Pierce Plates Detergent 2 plates PI88304 7K MWCO, 0.5 mL 40K MWCO, 2 mL 40–400 µL resin beds Sufficient for 25 samples, each 30–130 µL Sufficient for 25 samples, each 200–900 µL Removal 96-well Spin Plates Pierce Centrifuge Columns, 0.8mL 4 x 50 columns PI89869 Zeba Spin Desalting Columns, 50 columns PI89883 Zeba Spin Desalting Columns, 5 columns PI87770 Pierce Detergent Removal 25 columns PI87776 Polyethylene filter (30 µm pore size); screw-top caps Spin Columns, 125 μL 7K MWCO, 0.5 mL 40K MWCO, 5 mL Sufficient for microcentrifuge procedures with Sufficient for 50 samples, each 30–130 µL Sufficient for 5 samples, each 300–2,000 µL 40–400 µL resin beds Pierce Detergent Removal 25 columns PI87777 Zeba Spin Desalting Columns, 5 columns PI89889 Zeba Spin Desalting Columns, 25 columns PI87771 Pierce Centrifuge Columns, 2 mL 25 columns PI89896 Spin Columns, 0.5 mL 7K MWCO, 2 mL 40K MWCO, 5 mL Polyethylene filter (30 µm pore size); screw-top and Pierce Detergent Removal 5 columns PI87778 Sufficient for 5 samples, each 200–700 µL Sufficient for 25 samples, each 300–2,000 µL press-on bottom caps Spin Columns, 2 mL Zeba Spin Desalting Columns, 25 columns PI89890 Zeba Spin Desalting Columns, 5 columns PI87772 Sufficient for centrifuge procedures with 2 mL resin beds and 15 mL conical centrifuge tubes Pierce Detergent Removal 5 columns PI87779 7K MWCO, 2 mL 40K MWCO, 10 mL for collection Spin Columns, 4 mL Sufficient for 25 samples, each 200–700 µL Sufficient for 5 samples, each 1–4 mL Pierce Centrifuge Columns, 5 mL 25 columns PI89897 Pierce Detergent Removal Resin 10 mL PI87780 Zeba Spin Desalting Columns, 5 columns PI89891 Zeba Spin Desalting Columns, 25 columns PI87773 Polyethylene filter (30 µm pore size); screw-top and 7K MWCO, 5 mL 40K MWCO, 10 mL press-on bottom caps Sufficient for 5 samples, each 500–2,000 µL Sufficient for 25 samples, each 1–4 mL Sufficient for centrifuge procedures with 5 mL Learn more at thermofisher.com/detergentremoval Zeba Spin Desalting Columns, 25 columns PI89892 Zeba 96-well Spin Desalting Plates, 2 plates PI87774 resin beds and 15 mL conical centrifuge tubes for 7K MWCO, 5 mL 40K MWCO collection Sufficient for 25 samples, each 500–2,000 µL Sufficient for 2 × 96 samples, each 20–100 µL Pierce Centrifuge Columns, 10 mL 25 columns PI89898 Zeba Spin Desalting Columns, 5 columns PI89893 Zeba 96-well Spin Desalting Plates, 4 plates PI87775 Polyethylene filter (30 µm pore size); screw-top and 7K MWCO, 10 mL 40K MWCO press-on bottom caps Sufficient for 5 samples, each 700 µL–4 mL Sufficient for 4 × 96 samples, each 20–100 µL Sufficient for centrifuge procedures with 10 mL resin beds and 50 mL conical centrifuge tubes Zeba Spin Desalting Columns, 25 columns PI89894 for collection 7K MWCO, 10 mL Pierce Column Extenders 10 extenders PI69707 Sufficient for 25 samples, each 700 µL–4 mL Related desalting products Cat. Nos. 89896, 89897, 89898 Zeba Spin Desalting Plates, 7K MWCO 2 plates PI89807 Sufficient for increasing column reservoir volumes by Dextran Desalting Columns, 5 columns PI43230 Sufficient for 2 × 96 samples, each 20–100 µL approx. 35 mL 5K MWCO, 5 mL Zeba Spin Desalting Plates, 7K MWCO 4 plates PI89808 Sufficient for 5 samples, each 0.25–1.5 mL Sufficient for 4 × 96 samples, each 20–100 µL Dextran Desalting Columns, 5 columns PI43233 Zeba Desalting Chromatography Cartridges, 5 cartridges PI89934 5K MWCO, 10 mL Learn more at thermofisher.com/desalting 7K MWCO, 1 mL Sufficient for 5 samples, each 0.5–3 mL Sufficient for samples requiring 1 mL of resin for separation Polyacrylamide Spin Desalting Columns, 25 columns PI89849 7K MWCO, 0.7 mL Zeba Desalting Chromatography Cartridges, 5 cartridges PI89935 Sufficient for 25 samples, each 30–120 µL 7K MWCO, 5 mL Sufficient for samples requiring 5 mL Polyacrylamide Spin Desalting Columns, 50 columns PI89862 of resin for separation 7K MWCO, 0.7 mL Sufficient for 50 samples, each 30–120 µL Zeba Micro Spin Desalting Columns, 25 columns PI87764 40K MWCO, 75 µL Polyacrylamide Desalting Columns, 5 columns PI43426 Sufficient for 25 samples, each 5–14 µL 1.8K MWCO, 5 mL Sufficient for 5 samples, each 0.25–1.25 mL Zeba Micro Spin Desalting Columns, 50 columns PI87765 40K MWCO, 75 µL Polyacrylamide Desalting Columns, 5 columns PI43243 Sufficient for 50 samples, each 5–14 µL 6K MWCO, 10 mL Sufficient for 5 samples, each 0.5–2.5 mL Zeba Spin Desalting Columns, 25 columns PI87766 40K MWCO, 0.5 mL Sufficient for 25 samples, each 70–200 µL

44 45 Ordering Information

Ordering information References

Product Quantity Cat. No. Product Quantity Cat. No. References for Slide-A-Lyzer products 1. Willsey GG et al. (2015) J Bacteriol 198:301–310. 18. Srinivasan K et al. (2015) J Biomol Screen 37. Fu M et al. (2014) Mol Cancer Ther 13:902–915. 20:768–778. Protein concentration products Endotoxin removal and quantitation 2. Brown K et al. (2016) Hum Mol Genet 25:558–570. 38. Jessen DL et al. (2014) Antimicrob Agents Chemother 3. Vandavasi VG et al. (2016) Plant Physiol 170:123–135. 19. Chatalic KL et al. (2015) J Nucl Med 56:1094–1099. 58:839–850. Pierce Protein Concentrators PES, 25 units PI88512 Pierce High Capacity Endotoxin 5 columns PI88273 20. Kottom TJ et al. (2015) Infect Immun 83: 39. Tavaré R et al. (2014) PNAS 111:1108–1113. 3K MWCO, 0.5 mL Removal Spin Columns, 0.25 mL 4. Martínez-Cerdeño V et al. (2016) Cereb Cortex 26:374–383. 2816–2826. 40. Stensen MH et al. (2014) Hum Reprod 29:125–134. Pierce Protein Concentrators PES, 10 units PI88514 Pierce High Capacity Endotoxin 5 columns PI88274 21. Tang H et al. (2015) 3K MWCO, 2-6 mL Removal Spin Columns, 0.5 mL 5. Rwei AY et al. (2015) PNAS 112:15719–15724. Mol Cell Proteomics 41. Rajagopal BS et al. (2013) Biochem J 456:441–452. 14:1323–1333. Pierce Protein Concentrators PES, 24 units PI88515 Pierce High Capacity Endotoxin 25 columns PI88275 6. Chattopadhyay M et al. (2015) J Biol Chem 42. Ye X et al. (2013) Am J Physiol Endocrinol Metab 3K MWCO, 2-6 mL Removal Spin Columns, 0.5 mL 290:30624–30636. 22. Alghamdi AJ et al. (2015) Biol Reprod 92:94. 305:E1375–E1383. Pierce Protein Concentrators PES, 10 units PI88525 Pierce High Capacity Endotoxin 5 columns PI88276 7. Fei J et al. (2015) Genes Dev 29:2563–2575. 23. Kim JS et al. (2015) Infect Immun 83:1556–1567. 43. Cao Z et al. (2013) Mol Cell Proteomics 3K MWCO, 5-20 mL Removal Spin Columns, 1 mL 8. Vance DJ et al. (2015) Clin Vaccine Immunol 24. Sasso O et al. (2015) FASEB J 29:2616–2627. 12:2724–2734. Pierce Protein Concentrators PES, 24 units PI88526 Pierce High Capacity Endotoxin 25 columns PI88277 22:1285–1293. 25. Koudelka S et al. (2015) Stroke 46:ATP99. 44. Neguembor MV et al. (2013) J Mol Cell Biol 3K MWCO, 5-20 mL Removal Spin Columns, 1 mL 9. Paradela-Dobarro B et al. (2015) J Mol Endocrinol 26. Harmsen S et al. (2015) Sci Transl Med 7:271ra7. 5:294–307. Pierce Protein Concentrators PES, 4 units PI88534 Pierce High Capacity Endotoxin 10 mL PI88270 56:23 –37. 45. Heymans S et al. (2013) Circulation 128:1420–1432. 5K MWCO, 20-100 mL Removal Resin 27. Brunton PJ et al. (2015) J Neurosci 35:666–677. Pierce Protein Concentrators PES, 25 units PI88513 Pierce High Capacity Endotoxin 100 mL PI88271 10. Ponnusamy R et al. (2015) Nucleic Acids Res 28. Planagumà J et al. (2015) Brain 138:94–109. 43:10039–10054. 10K MWCO 0.5 mL Removal Resin 29. Fida TT et al. (2014) Appl Envir Microbiol Pierce Protein Concentrators PES, 10 units PI88516 Pierce High Capacity Endotoxin 250 mL PI88272 11. Linz LB et al. (2015) J Virol 89:11203–11212. 80:7725–7731. 10K MWCO, 2-6 mL Removal Resin 12. Vitrac H et al. (2015) PNAS 112:13874–13879. 30. Critchfield AS et al. (2014)Reprod Sci 21:1266–1273. Pierce Protein Concentrators PES, 24 units PI88517 Pierce LAL Chromogenic Endotoxin Kit PI88282 13. Bavagnoli L et al. (2015) Nucleic Acids Res 31. Moonah S et al. (2014) Eukaryot Cell 13:1337–1345. 10K MWCO, 2-6 mL Quantitation Kit, 50 tests 43:9405–9417. Pierce Protein Concentrators PES, 10 units PI88527 32. Oda Y et al. (2014) J Cell Sci 127:4201–4212. 14. Lee SJ et al. (2015) J Immunol 195:2282–2293. 10K MWCO, 5-20 mL 33. Seldin M et al. (2014) J Exp Biol 217:2667–2679. Learn more at thermofisher.com/endotoxinremoval 15. Klueh U et al. (2015) J Diabetes Sci Technol Pierce Protein Concentrators PES, 24 units PI88528 34. Thiele JR et al. (2014) Circulation 130:35–50. 10K MWCO, 5-20 mL 9:957–965. 35. Escudero CA et al. (2014) J Cell Sci 127:1966–1979. Pierce Protein Concentrators PES, 4 units PI88535 16. Bagley AF et al. (2015) Cancer Res 75:3255–3267. 36. Murphy JT et al. (2014) Blood 123:2172–2180. 10K MWCO, 20-100 mL 17. Ho R et al. (2015) Mol Biol Cell 26:2655–2663. Pierce Protein Concentrators PES, 25 units PI88502 30K MWCO, 0.5 mL Pierce Protein Concentrators PES, 10 units PI88521 References using Zeba desalting products 30K MWCO, 2-6 mL 1. Schwartz PA et al. (2014) PNAS 111:173–178. 19. Birk J et al. (2013) J Cell Sci 126:1604–1617. 39. Burgoyne JR et al. (2012) FASEB J 26:832–841. Pierce Protein Concentrators PES, 24 units PI88522 2. Wu J et al. (2013) J Biol Chem 288:35904–35912. 20. Labrijn AF et al. (2013) PNAS 110:5145–5150. 40. Antonucci F et al. (2012) J Neurosci 32:1989–2001. 30K MWCO, 2-6 mL 3. Issafras H (2013) J Pharmacol Exp Ther 21. Shi LX et al. (2013) PNAS 110:930–935. 41. Liu Y et al. (2011) J Nucl Med 52:1956–1963. Pierce Protein Concentrators PES, 10 units PI88529 348:202–215. 22. Kontos S et al. (2013) PNAS 110:E60–E68. 42. Ho AW et al. (2011) J Immunol 187:6011– 6021. 30K MWCO, 5-20 mL 4. Olsson N et al. (2013) Mol Cell Proteomics 23. Yang Y et al. (2013) Mol Cell Proteomics 12:237–244. 43. Zhang H et al. (2011) Mol Pharmacol 80:839 – 847. Pierce Protein Concentrators PES, 24 units PI88531 12:3612–3623. 30K MWCO, 5-20 mL 24. Peisley A et al. (2012) PNAS 109:E3340–E3349. 44. Liu H et al. (2011) PNAS 108:18536–18541. 5. Rascoe LN et al. (2013) Clin Vaccine Immunol Pierce Protein Concentrators PES, 4 units PI88536 25. Kappel L et al. (2012) J Cell Biol 199:771–782. 45. Olsson N et al. (2011) Mol Cell Proteomics 20:1758–1763. 30K MWCO, 20–100 mL 26. Vomaske J et al. (2012) J Virol 86:11833–11844. 10:M110.003962. Pierce Protein Concentrators PES, 25 units PI88503 6. Andacht TM et al. (2014) J Anal Toxicol 38:8–15. 27. Fan Y et al. (2012) Bioact Compat Polym 27:585–603. 46. Skipsey M et al. (2011) J Biol Chem 100K MWCO, 0.5 mL 7. Foo JY et al. (2013) Clin Chem 59:1523–1531. 286:32268–32276. 28. Xu S et al. (2012) PNAS 109:16348–16353. Pierce Protein Concentrators PES, 10 units PI88523 8. Kryndushkin D et al. (2013) J Biol Chem 47. Mazier S et al. (2011) J Biol Chem 100K MWCO, 2-6 mL 29. Bista M et al. (2012) PNAS 109:15752–15756. 288:27100–27111. 286:29347–29355. Pierce Protein Concentrators PES, 24 units PI88524 9. Petitdemange C et al. (2013) Clin Infect Dis 30. McKinney KQ et al. (2012) Cancer Genomics 100K MWCO, 2-6 mL 48. Lin AY et al. (2011) PNAS 108:12729–12733. 57:745–755. Proteomics 9:257–263. Pierce Protein Concentrators PES, 10 units PI88532 49. Goyal A et al. (2011) J Biol Chem 286:25947–25962. 10. Albrecht SC et al. (2014) J Biomol Screen 31. Suzuki Y et al. (2012) Plant Physiol 160:533–540. 100K MWCO, 5-20 mL 50. Vinegoni C et al. (2011) Sci Transl Med 3:84ra45. 19:379–386. 32. Qu L et al. (2012) J Neurosci 32:9554–9562. Pierce Protein Concentrators PES, 24 units PI88533 51. Murray CI et al. (2011) Mol Cell Proteomics 11. Gerdes MJ et al. (2013) 110:11982–11987. 33. DiGiandomenico A et al. (2012) J Exp Med 100K MWCO, 5-20 mL PNAS 10:M110.004721. 12. Palzer S et al. (2013) Eukaryot Cell 12:816–827. 209:1273–1287. 13. Persson H et al. (2013) Infect Immun 81:2236–2241. 34. Gavin JM et al. (2012) J Biol Chem Learn more at thermofisher.com/concentrators 287:15512–15522. 14. Haeussler DJ et al. (2013) J Biol Chem 288:15380–15389. 35. Madabhushi SR et al. (2012) Blood 119:4769–4778. 15. Gavrilyuk J et al. (2013) J Virol 87:4985–4993. 36. Rodríguez–Rubio L et al. (2012) Appl Environ Microbiol 78:2241–2248. 16. Sinz Q et al. (2013) Appl Environ Microbiol 79:2284–2293. 37. Žumer K et al. (2012) Mol Cell Biol 32:1354–1362. 17. Muretta JM et al. (2013) PNAS 110:7211–7216. 38. Woods RJ et al. (2012) Diabetes Sci Techno 6:265–276. 18. Frasier CR et al. (2013) Cardiovasc Res 98:47–55.

46 47 Download free copies of our protein research handbooks

Protein transfer technical handbook Protein research handbook This handbook is designed to provide you with what you need to know This handbook provides a technical overview of key methods in protein about the protein transfer process, from transfer systems to monitoring research, as well as guidance on choosing the right tools for optimal transfer efficiency. This handbook offers you a deep dive into different results. The brochure features sections focusing on expression, isolation transfer systems, technical data, protocols, and troubleshooting tips for and purification, electrophoresis, western blotting, mass spectrometry, Protein Research Handbook better protein transfer, and includes easy-to-use selection guides to help Expression • Isolation and purifi cation • Gel electrophoresis • assays, and crosslinking and modification. Western blotting • Assays and analysis • Mass spectrometry • Modifi cation and crosslinking you find the right products for your needs.

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For Research Use Only. Not for use in diagnostic procedures. © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Bio-Rad, Bio-Spin, Bio-Scale, Bio-Gel, and Affi-Prep are trademarks of Bio-Rad Laboratories Inc. Brij is a trademark of ICI America Inc. EndoTrap is a trademark of Hyglos GmbH. MASCOT is a trademark of Matrix Science. Millipore is a trademark of EMDMillipore. SpinTrap, HiTrap, Sephadex, MiniTrap, MidiTrap, ÄKTA FPLC are trademarks of GE Healthcare. Pall is a trademark of Pall Corp. ReductEtox is a trademark of Sterogene Bioseparations Inc. Sigma is a trademark of Sigma-Aldrich Co. LLC. BN0322162 0516