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Protein Clean-Up Technical Handbook Dialysis • Desalting • Detergent Removal • Concentration • Endotoxin Removal Contents Protein Clean-Up Technical Handbook

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Protein Clean-Up Technical Handbook Dialysis • Desalting • Detergent Removal • Concentration • Endotoxin Removal Contents Protein Clean-Up Technical Handbook 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,
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