Strep-Tag® -Broschure

Solutions iba For Life Sciences Strep-tag® -Broschure

Strep-tag®

Purification, Detection and Immobilization of Strep-tag® proteins with Strep-Tactin®

The protein production system for www.strep-tag.com highly pure proteins

www.iba-lifesciences.com THE STREP-TAG®- TECHNOLOGY

Strep-tag®II Strep-Tactin® 1. Strep-tag®II Only 8 amino acids Rapid one-step purification under physiological conditions High purity due to low tendency of Strep-Tactin® for unspecific protein binding Recombinant protein No tag removal necessary, N- and C-terminal fusion possible

2. Twin-Strep-tag® Higher affinity for Strep-Tactin®, same mild elution conditions More efficient capture of proteins from diluted cell culture supernatants

Twin-Strep-tag®

3. Strep-Tactin® Strep-Tactin® Purification of Strep-tag® and Twin-Strep-tag® proteins Detection of Strep-tag® and Twin-Strep-tag® proteins

4. NEW Strep-Tactin XT New developed variant for immobilization /assays Strep-Tactin XT pM affinity for Twin-Strep-tag® – tight binding Allows purification under denaturing conditions (6 M urea)

His-STREPPER 5. His-STREPPER Adapter molecule which converts His-tag fusion proteins into Strep-tag® fusion proteins 6x-His-tag No cloning required

Strep-Tactin® 6. Detection and Visualization Strep-Tactin® and Strep-tag® antibodies for detection Fluorescent conjugates HRP and AP conjugates Strep-tag®-antibody

7. Immobilization Twin-Strep-tag® binds to Strep-Tactin XT with an affinity in pM range - very low off-rate but mild elution still possible

2 CONTENT

1. Technologies...... 4

1.1 Strep-tag®II...... 4

1.2 Twin-Strep-tag®...... 6 1.3 Strep-Tactin XT...... 7 1.4 His-STREPPER...... 8

2. Products...... 8

2.1 Expression of (Twin-) Strep-tag proteins...... 8

2.2 Purification of (Twin-)Strep -tag proteins...... 9 Strep-tag®II – Starter Kits...... 9 Twin-Strep-tag® – Starter Kits...... 10 WET-FRED ...... 10 Buffers and Reagents...... 11 Strep-Tactin® Resins...... 12 MagStrep: Strep-Tactin coated Magnetic Beads...... 12 Columns, cartridges, plate formats and adapters...... 13

2.3 Detection of (Twin-) Strep-tag proteins...... 14

2.4 Immobilization of (Twin-) Strep-tag proteins...... 16

2.5 Strep-/ His-tag products for protein purification...... 18

3. Ten Reasons to buy Strep-tag®...... backside

Why we use Strep-tag®!

“Why we admire the Strep®-tag purification system: (i) generally good expression of recombinant proteins, (ii) simplicity of protein purification and very high purity of isolated proteins (the latter is very important for crystallographic projects!), (iii) robust system to generate, reliably purify, and biochemically analyze various mutant derivatives reproducibly.” Prof. Dr. Erhard Bremer, Laboratory for Microbiology, Department of Biology, Philipps University Marburg, Germany.

“The Strep II tag appears to be an excellent candidate for affinity purification in general since it is a short tag that produces high purity material in good yields at a moderate cost. / ... / we find that a combination of His-tag and StrepII tag allows rapid capture of the tagged protein or protein complex from crude extracts...” Lichty et al. 2005, Protein Expression and Purification 41: 98-105.

3 1. Technologies

1. TECHNOLOGIES

1.1 STREP-TAG®

For the use of this system we offer Facts about Strep-tag® a large range of high quality ® ® products, such as The Strep-tag /Strep-Tactin system is one of the most widely used affinity chromatography systems in protein purification, • Cloning/Expression Vectors for detection and immobilization. different hosts • Various Starter Kits for newcomers Advantages: Highly pure proteins due to specific binding • Different Strep-Tactin® purificationresins and Biological function of proteins is preserved pre-packed columns ® • Detection Systems with The Strep-tag is a short peptide consisting of 8 amino acids. It Strep-Tactin® conjugates or a was initially developed for the specific reversible binding to the monoclonal antibody biotin pocket of streptavidin. Streptavidin was later on engineered to obtain the highly selective Strep-Tactin®, which binds the Strep-tag®II with a nearly 100 times higher affinity compared to streptavidin. The Strep-tag®II can be fused to the protein as either N- or C-ter- streptavidin minal tag. Physiological buffers in combination with a wide range of additives can be used. The elution is performed competitively

biotin with desthiobiotin, an inexpensive, reversibly binding and stable analog of biotin.

Strep-tag® Protein Purification System The Strep-tag® protein purification system provides the reliable Strep-tag® is a peptide that was engineered for the specific binding one-step purification of proteins suitable for a broad range of ap- to the biotin binding pocket of plications, including e.g.: Streptavidin. Structural and functional investigations Crystallization for determination of 3D structure Strep-tag® II – the short 8-amino acid tag does not influence the protein. Due to the mild conditions Strep-tag® II recombinant proteins can

NH2-WSHPQFEK-COOH also be used for: Assays involving protein-protein and protein-DNA interactions Investigating ligand-receptor interactions under physiological conditions Separating living cells for re-culturing purposes Strep-tag®II ® Strep-Tactin Strep-tag is the method of choice for multiple protein classes: Metalloproteins / Enzymes Membrane proteins Sensitive protein complexes with multiple subunits And any other protein Recombinant protein

Recombinant protein with Strep-tag®II bound to Strep-Tactin.

4 1.Technologies

Strep-tag® Principle Binding Purification procedure under physiological conditions ƌĞĐŽŵďŝŶĂŶƚ 1. ƉƌŽƚĞŝŶ ^ƚƌĞƉͲdĂĐƟŶΠ The purification ofStrep -tag®II fusion proteins is easy, straight- forward and user-friendly. The complete procedure should be ŚŽƐƚƉƌŽƚĞŝŶƐ ĞƐŝŶ performed under physiological conditions at a pH >7.0. ƌ ^ƚƌĞƉͲƚĂŐ//

Steps 1 + 2: The cell lysate is subjected to the column. Once the 2. tagged protein has bound specifically toStrep -Tactin® the host proteins are rapidly washed off due to addition of low amounts of physiological wash buffer (Buffer W).

Step 3: Then, bound Strep-tag®II protein is gently eluted by addition of wash buffer supplemented with 2.5 mM desthiobiotin Elution (Buffer E), which specifically competes for the biotin binding ĞƐƚŚŝŽďŝŽƟŶ pocket. 3. Since the buffer conditions during elution essentially remain unchanged, unspecific bound proteins (withoutStrep -tag®II) will not be eluted and, thus, will not contaminate the protein of interest. Besides the specific binding ofStrep -tag® to Strep-Tactin®, this is the second specificity conferring step of the purification procedure. Due to the competitive binding, only Strep-tag® fusion protein will be eluted, yielding extremely high protein purity.

Regeneration Steps 4: In order to regenerate the column the yellow azo dye HABA (2- [4’-hydroxy-benzeneazo] benzoic acid) is added as 4. component of buffer R in excess to displace desthiobiotin from the HABA (bound) binding pocket. Once HABA binds to the binding site, the color turns to red conveniently indicating the regeneration and activity status of the column.

Step 5: HABA can be removed simply by adding wash buffer. 5. Once the red color disappeared the column can be re-used. ,;ŶŽƚďŽƵŶĚͿ Strep-Tactin® resin can be regenerated and re-used 3 to 5 times without loss in performance.

Strep-Tactin® columns regenerated with HABA (Sepharose, Superflow, Macroprep from left to right)

5 1. Technologies

1.2 TWIN-STREP-TAG®

Twin-Strep-tag® The high affinity tag for Protein Complex Purification and Protein:Protein Interaction Analysis Based on the proprietary Strep-tag® technology the Twin-Strep-tag® a sequential arrangement of two Strep-tag®II was developed. This tag enables the same mild and rapid purification asStrep -tag®II, but it has a significantly increased affinity forStrep -Tactin® and a better availability because of its Twin-Strep-tag®: The high affinity, larger size. tandem Strep-tag® for Protein Complex Purification and Protein:Protein Interac- Benefits of the Twin-Strep-tag® tion Analysis Twin-Strep-tag® has a neutral amino acid composition Twin-Strep-tag®: tandem arrangement > 95 % pure protein after a single purification step of two Strep-tag®II (total size of 30 aa) High affinity leads to good protein yields Specific competitive elution with small amounts of (desthio-) SA-WSHPQFEKGGGSGGGSGGSA biotin in the physiological wash buffer WSHPQFEK Tolerates elevated levels of additives and detergents

Twin-Strep-tag® is very well suited for the following applications Twin-Strep-tag® is the tag of choice for highly diluted target proteins, Protein:Protein Interaction Analysis e.g. from cell culture supernatants. Protein Complex Isolation Purification of highly diluted proteins from e.g. cell culture supernatants

Twin-Strep-tag® should be used for proteins hampering the More information can be obtained from binding ability of Strep-tag®II to ® Strep-Tactin . Due to its tandem Schmidt et al., 2013 arrangement of two Strep-tag®II, “Development of the Twin-Strep-tag® and its application the Twin-Strep-tag® overcomes negative influences of the target for purification of recombinant proteins from cell culture protein. supernatants” Protein Expression and Purification; Volume 92, Issue 1

Protein:Protein Interaction Analysis The high specificity of the Twin-Strep/Strep-Tactin® interaction and ® Twin-Strep-tag® low tendency of Strep-Tactin to bind proteins non-specifically

preys 1- 5 makes the system very attractive for protein:protein interaction

2 1 analysis.

3 bait Superﬂow® Features: 4 6 5 Better availibility of the tag due to the sequential arrangement Strep-Tactin® protein 6 of two Strep-tag®II High purity after one purification step High specificity - low tendency for false positive binding

6 1.Technologies

Twin-Strep-tag and Strep-Tactin XT for immobilization and assays As a peptide tag Twin-Strep-tag® The development of Strep-Tactin XT (see next chapter) extends the is especially used in purification use of Twin-Strep-tag® to the field of assay applications. of protein complexes, given that its size has nearly no influence on the complex formation in vivo Features: compared to protein tags, like MBP ® pM affinity of Twin-Strep-tag to Strep-Tactin XT – stable or GST. binding Reversibility of Twin-Strep/Strep-Tactin XT binding High specificity- low background Stable under physiological conditions Allows usage of detergents and additives

1.3 STREP-TACTIN XT

Strep-tag® conquers the world of assay implementation In order to overcome certain limitations of the Strep-tag® technology with respect to binding stability, in the field of assay usage the binding affinity ofStrep -tag®II to Strep-Tactin® had to be improved. Therefore, Strep-Tactin XT was engineered. Strep-Tactin XT has a binding affinity in double digit pM range for Twin-Strep-tag® and in nM range for Strep-tag®II. This, improve- ment in binding stability especially for Twin-Strep-tag® makes the Strep-tag® technology amenable to assay applications. Strep-Tactin XT

Advantages of Strep-Tactin XT: Strep-Tactin XT Edition: Allows the immobilization in very stable ranges (T1/2 = 13 h) The interaction with (Twin-) Strep-tag is still reversible All products conjugated with the new high affinity Strep-Tactin XT Improves the performance of the Strep-tag® technology are labelled with XT in the product in assay s name. Purification even under denaturing conditions (6 M urea) possible

When to use which TAG with which STREP-TACTIN® RESIN?

Strep-Tactin® Strep-Tactin XT

Application and Conditions Strep-tag®II Twin-Strep-tag® Strep-tag®II Twin-Strep-tag®

Concentrated (cytosolic) 4 4 4 4

Diluted (secretion) 4 4 4 4 Purification Batch (magnetic beads) 8 4 4 4

Denatured (6M urea) 8 8 4 4

Detection (Western, ELISA) 4 4 4 4

Assay/immobil. (MTP, chips) 8 4 4 4

7 1. Technologies

1.4 His-STREPPER Tris-NTA Strep-tag®II Strep your His-tag without cloning His-STREPPER adapter molecule converts a 6xHis-tag fusion protein into a Strep-tag®II carrying protein. The molecule comprising Strep-tag®II (SA-WSHPQFEK) conjugated with a nickel charged. It tightly binds to 6xHis-tag and thereby converts a 6xHis-tag fusion protein to a Strep-tag®II fusion protein without the need for clon- ® His-STREPPER ing. It gives access to the advantages of the Strep-tag purification system, namely the high purity of the isolated proteins, without a time-consuming cloning process to His-tag users. (More information on page 19)

Features: His-STREPPER conveys the Adapter molecule for fast and easy conversion of benefits of theStrep -tag® ® Technology to 6xHistidine fusion 6xHistidine-tag fusion protein to Strep-tag II fusion proteins proteins. Transfer all Strep-tag®II advantages (pure & functional proteins) to 6xHis-tag proteins Cost- and time-effective

His-STREPPER purifies 6xHis fusion proteins to higher purity Purification results for GFP-His using different purification protocols. GFP-His was either purified using the His-STREPPER and Strep-Tactin® in PBS pH 8 buffer or using Ni-NTA under the same physiological or non-physiological conditions. His- STREPPER:Strep-Tactin provides better results than His-tag:Ni-NTA.

Crude lysate + Ni-NTA-Superflow + His-STREPPER + Strep-Tactin-Superflow

Purity = 44% Purity = 99%

2. PRODUCTS

2.1 EXPRESSION

® ® Combinatorial Sites A For expression of Strep-tag II and Twin-Strep-tag fusion proteins tet f1 o ri IBA offers a variety of expression vectors (StarGate Cloning

ri o l System) lE o C For E. coli, mammalia, insect cells and yeast pASG-IBA Strep-tag®, Twin-Strep-tag®, His-tag, GST-tag and FLAG-tag vectors

te t re p R re p ss Am More information can be found on our homepage or www.stargate-cloning.com

8 2. Products

Furthermore the MEXi® (Mammalian Expression IBA) system was MEXi® Mammalian Expression IBA developed to provide an optimized system for expression of Twin-/ system: Strep-tag®II fusion proteins in mammalian HEK293 cells. It comes along with a HEK293E cell line (MEXi® 293E), a transfec- • High protein yields tion (MEXi®-TM) and cultivation (MEXi®-CM) medium and an • Easy handling optimized vector system (pDSG-IBA). • Optimized components • Cost-effective

2.2 PURIFICATION

STREP-TAG®II STARTER KITS Attractive offers for newcomers and experts are our Strep-tag®II Starter Kits containing all essential reagents required for expression in E. coli, purification and detection ofStrep -tag®II proteins.

All Strep-tag®II Starter Kits include: Control plasmid with a 15 kD protein insert Anhydrotetracycline for induction of expression Fractionation buffer for the preparation of a periplasmic extract Strep-tag-Starter Kit with one Wash buffer for column chromatography or for the preparation purification column of a cytoplasmic extract Elution buffer for displacing the Strep-tag®II protein from the column Column regeneration buffer (with HABA) Strep-Tactin® horse radish peroxidase (HRP) conjugate for Western blot detection.

In addition the different Starter Kits are equipped with different columns or cartridges to enable the evaluation of the optimal resin for a particular protein of interest:

Strep-tag® Starter Kit Contains one ready-to-use column with Strep-Tactin® Sepharose®. Cat. no. 2-1101-000

Strep-tag® Starter Kit 3C Includes 3 different columns with Strep-Tactin® immobilized to Sepharose®, MacroPrep® and Superflow®, respectively, allowing the evaluation of the optimal resin for your particular protein of interest. Cat. no. 2-1102-001

Strep-well HT 50 Purification Starter Kit The perfect solution for automated, high-throughput purification of Strep-tag® proteins. Allows simultaneous purification of up to 96 different Strep-tag® proteins and yields in up to 200 µg highly pure Strep-tag® protein per well. Cat.no: 2-1701-000

9 2. Products

TWIN-STREP-TAG® - STARTER KIT

For Twin-Strep-tag® newcomers we provide kits for cloning and for Further methods for PPI analysis are purification respectively. our tandem affinity purification kits: • One-TAP StarGate Twin-Strep Cloning Kit, E. coli (5-1621-001) - With Twin-Strep-tag only StarGate Twin-Strep Cloning Kit, mammalia (5-1621-002) • Two-TAP Twin-Strep Basic Purification Kit (2-1121-010) - Using two affinity tags (Twin-Strep-tag/FLAG-tag The Cloning Kit provides all necessary components for cloning a For both is a Cloning Kit, for either certain gene of interest (GOI) into Twin-Strep-tag® vectors and the mammalian cells or E. coli available Basic Purification Kit consists of all components required for a first purification trial. Note, that the components of the Basic Purifica- tion Kit are adjusted to the protein:protein interaction procedure where a single-use of Strep-Tactin® columns is recommended.

For protein:protein interaction analysis in combination with Twin-Strep-tag® further methods are described. Detailed information about our protein:protein interaction technologies and products are available on our homepage (www.iba-lifesciences.com).

WET FRED

(Twin-) Strep-tag® purification from cell culture supernatant In mammalian and insect cell expression recombinant proteins are often secreted in the cell culture medium. Thus, the protein is present in large volumes before the purification step. To apply this Components of large volume onto a gravity flow column with a certain flow the WET FRED WET FRED applicator was developed. This device facilitates the transfer of large volumes to a Strep-Tactin® gravity flow column for purification of the recombinant target protein fused with (Twin-) Strep-tag®. It works simply by hydrostatic pressure (siphon principle). Due to its small size and flexibility it is easy to handle at the bench, in the cold room or in the fridge. Columns cannot run dry and need no supervision. Furthermore, no sophisticated software is necessary facilitating set up and use.

height to be adjusted for Cat.no. 2-0911-001 for 1ml columns and 2-0910-001 for 5-10ml ﬂow regulation columns

Experimental set-up

10 2. Products

BUFFERS AND REAGENTS

Product Contents Cat.no Avidin (high grade for PPI applications) 15 mg 2-0204-015 50 mg 2-0204-050 Anhydrotetracycline 25 mg 2-0401-002 (inducer for tet promotor) 50 mg 2-0401-001 BioLock biotin blocking solution 50 ml 2-0205-050 250 ml 2-0205-250 Strep-tag® protein purification buffer set 100 ml 10x Buffer W 2-1002-001 25 ml 10x Buffer E 100 ml 10x Buffer R D-Desthiobiotin 1 g, 2-1000-002 5 g 2-1000-005 Strep-Tactin® columns regenerated with Elution Buffer with D-Desthiobiotin 25 ml 2-1000-025 HABA (see also Strep-tag® purification (10x Buffer E) cycle): Strep-tag® regeneration buffer with 100 ml 2-1002-100 Strep-Tactin® Sepharose®, Superflow® HABA (10x Buffer R) and MacroPrep® (left to right). Strep-tag® washing buffer (10x Buffer W) 100 ml 2-1003-100 Since MacroPrep® is not as transparent Strep-tag®II Peptide 1.8 mg 2-1018-002 as Sepharose® or Superflow®, the color Biotin Elution Buffer (10x Buffer BE) 25 ml 2-1019-025 shift to red is less visible. Biotin Elution Buffer (5x Buffer BX) for 50 ml 2-1040-050 MacStrep “type3” XT

BioLock biotin blocking solution Culture media often contain significant amounts of biotin. This is especially the case for mammalian or insect cell culture media. When proteins from biotin containing extracts or media are inten- ded to be purified viaStrep -Tactin® chromatography, biotin must be masked by the addition of avidin prior to the application onto d-biotin the column. BioLock biotin blocking solution allows this masking of biotin (and biotinylated proteins) in a very fast and convenient way. Activity: >70U/ml; add at least 1U of BioLock solution per µg of biotin. Cat.no. 2-0205-050

Biotin Elution Buffer BX The new Strep-TactinXT variant requires a more competitive elution buffer due to the higher affinity of Strep-Tactin XT for Strep-tag®II and especially Twin-Strep-tag®. Biotin Elution Buffer BX has a higher biotin concentration to enable an efficient elution from e.g. MagStrep “type3” XT beads. Cat.no. 2-1040-050

11 2. Products

STREP-TACTIN® RESINS

Different types of Strep-Tactin® resins are provided ® Which Resin to use? Several Strep-Tactin resin versions are available which differ in Test our different Strep-Tactin® their properties and suitability for applications. resins to evaluate the best one for ® ® the purification of your protein of Strep-Tactin Sepharose is preferentially used for gravity flow interest. The Strep-tag® Starter Kit chromatography. 3C (2-1102-001) consits of Strep-Tactin® Superflow®, can also be used for low pressure, FPLC and 1 ml gravity flow columns of Strep-Tactin® Superflow® High HPLC applications. In addition, Superflow Capacity (HC) Strep-Tactin®-Sepharose®, is especially suited for increased flow rates Strep-Tactin® MacroPrep® and for the purification of large protein com- ® ® -Superflow and -MacroPrep , plexes (see: www.strep-tag.com). respectively. MagStrep Beads (Strep-Tactin® XT for batch purification only. coated magnetic Beads)

MAGSTREP:STREP-TACTIN® COATED MAGNETIC BEADS

MagStrep (Strep-Tactin XT coated Magnetic Beads) “type3” XT enable the fast purification ofStrep -tag® fusion proteins in batch formate. In addition, it allows purification from small volumes.

Features: High binding capacity (1-3 nmol/µl beads, corresponding to 30-90 µg of a 30 kDa protein) Very low non-specific protein binding due to improved coating Flexible elution conditions, under denaturing conditions by boiling in SDS gel loading buffer or under native conditions with biotin High affinity for Twin-/Strep-tag®II – due to new Strep-Tactin XT

MagStrep “type3” XT beads: Cat.no 2-4090-002 Magnetic separator: Cat.no 2-1602-000 Biotin Elution Buffer (5x Buffer BX): Cat.no 2-1040-050 Crude bacterial extract before Native purification using Denaturing purification using sample purification buffer BX for elution buffer and boiling for elution

-tag -tag

-tag Strep Strep

Strep GFP- GFP-

GFP-

Purity > 95% -Tactin XT Purity > 95%

Strep

Fig: Purification of GFP-Strep-tag fusion protein from crude bacterielle extract using MagStrep “type3“ XT beads. Due to specific binding properties of the beads even the elution by boiling leads to highly pure proteins. (Protein purification analysis was performed with an Agilent Bioanalyzer 2100 system instead of SDS-PAGE)

12 2. Products

Different Strep-Tactin® Resin Specifications

Strep-Tactin® Strep-Tactin® Strep-Tactin® Strep-Tactin® MagStrep Sepharose Superflow Superflow MacroPrep Magnetic Beads high capacity Gravity flow column Yes Yes Yes Yes No Batch purification For Twin-Strep-tag For Twin-Strep-tag For Twin-Strep-tag For Twin-Strep-tag Yes* only only only only FPLC No Yes Yes Yes No HPLC No Yes Yes Yes No Binding capacity 50 - 100 nmol/ml 50 - 100 nmol/ml 150 - 500 nmol/ml 50 - 100 nmol/ml Type3: 1 - 3 nmol/µl beads Support Sepharose 4FF Superflow 6 Superflow 6 MacroPrep 50 / Bead structure agarose 6 % agarose, 6 % agarose, polymethacrylate crosslinked crosslinked Bead size 45-165 µm 60 - 160 µm 60 - 160 µm 50 µm Type3: 25 µm spherical spherical Exclusion limit 3 x 107 6 x 106 6 x 106 1 x 106 / Recommended up to 30 cm/h up to 300 cm/h up to 300 cm/h up to 300 cm/h / linear flow rate pH stability 4 - 9 2 - 13 2 - 13 2 - 12 n.d. Max. pressure Gravity flow 9.6 bar (cartridge 9.6 bar (cartridge 70 bar (cartridge / cover 3 bar only) cover 3 bar only) cover 3 bar only)

* For Strep-tag®II only recommended if expression rate >1 mg/ml otherwise use Twin-Strep-tag®

COLUMNS, CARTRIDGES, PLATE FORMATS AND ADAPTERS

No Description Application Volume Strep-Tactin® Strep-Tactin® Strep-Tactin® Strep-Tactin® applied Sepharose Superflow Superflow HC MacroPrep 1 0.2 ml gravity flow Gravity flow 0.1 – 2 ml 2-1202-550 2-1207-550 2-1209-550 2-1506-550 columns (5 columns) 2 1 ml gravity flow column Gravity flow 0.5 – 10 ml 2-1202-001 2-1207-001 2-1209-001 2-1506-001 3 5 ml gravity flow column Gravity flow 2.5 – 50 ml 2-1202-051 2-1207-051 2-1209-051 2-1506-051 3 10 ml gravity flow column Gravity flow 5 – 100 ml 2-1202-101 2-1207-101 2-1209-101 2-1506-101 4 1 ml cartrigde FPLC/HPLC 0.5 – 10 ml – 2-1235-001 2-1237-001 2-1537-001 4 5 ml cartrigde FPLC/HPLC 2.5 – 50 ml – 2-1236-001 2-1238-001 2-1538-001 5 Spin Columns Spinning Up to – – – 2-1850-050 (for 150 µg protein each) 500 µl Kit: 2-1800-000, 2-1850-010 6 Strep-Well HT 50 plates High 50 µl – 1 ml – – – 2-1725-010 (10 x 96well plates, for throughput/ Kit: 100 µg protein/well) Vacuum based 2-1700-000

Adapters for H-PR cartrigdes 1 2 3 6

4 5 Syringe 1/16 inch M6 adapter 1/4-28 Coupling adapter set adapter set set for GE adapter set adapter set (Luer-lock) for peristaltic Healthcare for FPLC for connecting Cat. No. pump tubing FPLC other other than GE up to three 2-1021-001 Cat. No. than ÄKTATM Healthcare cartridges 2-1025-001 Cat. No. Cat. No. Cat. No. 2-1022-001 2-1023-001 2-1026-001

13 2. Products

2.3 TWIN-/STREP-TAG® PROTEIN DETECTION SYSTEM

The Strep-tag® protein detection system supports a broad variety The following monoclonal anti- of assays, including: bodies against Strep-tag® are available: Western blot and ELISA procedures (colony blot, dot blot) • StrepMAB-Classic, unconjugated Screening for positive expression clones • StrepMAB-Classic HRP Monitoring expression levels and stability of Strep-tag® conjugate (HRP, horseradish peroxidase) proteins • StrepMAB-Immo - the high Immunocytochemistry and immunohistochemistry affinity antibody for capturing Protein localization and targeting studies Strep-tag® proteins on solid surfaces Detection of Strep-tag® fusion proteins in Western blots/ELISA For direct detection of Strep-tag® fusion proteins in Western blots via chemiluminescence reaction IBA offers: Strep-Tactin® HRP conjugate (HRP, horseradish peroxidase) StrepMAB-Classic HRP conjugate. (no secondary antibody is needed!)

For direct detection of Strep-tag® fusion proteins in Western blots Direct detection of recombinant via chromogenic reaction use ® Strep-tag II GFP in a Western blot Strep-Tactin® AP conjugate (AP, alkaline phosphatase) using Strep-Tactin® HRP conjugate Strep-Tactin® HRP conjugate (HRP, horseradish peroxidase)

For protein detection in ELISA use 100

80 StrepMAB-Classic, unconjugated FACS ® x

a 60 Strep-Tactin HRP conjugate M f o analysis of

% ® 40 Strep-Tactin AP conjugate Zymosan, 20 stained

0 0 10 2 10 3 10 4 10 5 with APC-A StrepMAB Detection of Strep-tag® fusion proteins in Zymosan_Unstained.fcs 21.1 Immo Zymosan_C MAB immo O645.fcs 43.6 Immunofluorescence/FACS Zymosan_Clec7 MAB immo O645.fcs 2581 Oyster 645 For the detection of Strep-tag® fusion proteins in Immunofluores- against cense and FACS use Clec7-One-STrEP-tag 1:100, 20 min ice Kindly provided by: Dr. K. Neumann, III StrepMAB-Classic, unconjugated Med. Klinik, TUM München StrepMAB-Immo, unconjugated

Or directly conjugated Strep-Tactin®, Immunofluores- cence of type-I StrepMAB-Classic transmembrane StrepMAB-Immo protein (extracel- labeled with Chromeo™ 488, Chromeo™ 546 or Oyster® 645 lular domain respectively. N-terminal Strep- tagged) stained with Strep-Tactin® Chromeo 546 1:100 in insect cells. Fixation: 4% Formalde- hyd in PBS, 20minuten RTKindly provided by: Dr. Michael Krahn, , Abteilung Stammzellbiologie, GZMB Göttingen

14 2. Products

Two different detection systems are available to detect Strep-tag®II as well as Twin-Strep-tag® at the N-terminus,

C-terminus: Strep-Tactin® a) Monoclonal antibodies (mAB) , conjugated and unconjugated ® b) Strep-Tactin conjugates Strep-tag®-antibody

Product overview:

Detection a) Monoclonal antibodies b) Strep-Tactin® conjugates System StrepMAB- StrepMAB- Strep-Tactin® Strep-Tactin® Classic Classic HRP HRP conjugate AP conjugate conjugate Description Strep-tag®II- Strep-tag®II- Strep-Tactin® Strep-Tactin® and and protein, protein, Twin-Strep-tag®- Twin-Strep-tag®- labeled with labeled with specific mono- specific mono- horse radish alkaline clonal antibody, clonal antibody, peroxidase phosphtase unlabeled labeled with horse radish peroxidase Features - Highly - Highly - Sensitive - Very sensitive selective selective - Fast detection - Fast detection - Low back- - Low back- protocols protocols ground ground Secondary Yes, secondary No, direct No, direct No, direct The Strep-tag® Protein Ladder is antibody anti-mouse IgG, detection via detection via detection via designed for accurate MW determina- required HRP-conjuga- HRP HRP AP (cat. no. ted, required tion on Coomassie Blue stained gels 2-1591-001) and as positive control on Western Western blot, Suited, but not Recommended Recommended Recommended blots. As each protein contains the chromogenic recommended Strep-tag®II sequence which is detection detected by our Strep-Tactin® conju- Western blot, Not Recommended Recommended Not determined gates or Strep-tag® specific antibodies, chemilumi- recommended nescent the ladder can also be used for MW detection determinations on Western blots and (ECL) serves as a positive control for the Immuno- Recommended For ELISA only For ELISA only For ELISA only various detection systems. fluorescence, ELISA, FACS* Cat.No. 2-1011-100 Detects also – – + + biotinylated proteins Cat. no. 2-1507-001 2-1509-001 2-1502-001 2-1503-001 (100µg) (75µg for25 - 30 (0.5 ml) (0.5 ml) Western Blots) Available as – – 2-1502-000 2-1503-000 complete kit with all reagents required

Fluorescent conjugates: mAb and Fab for different spe- cies available: Labelling StrepMAB-Classic StrepMAB-Immo Strep-Tactin® StrepMAB Classic and StrepMAB Immo are available as full length 50 µg amounts 50 µg amounts 50 µg amounts · Chromeo™ 488 2-1544-050 2-1546-050 2-1542-050 antibodies and Fab for human, · Chromeo™ 546 2-1550-050 2-1552-050 2-1548-050 mouse, rat or rabbit. · Oyster®-645 2-1555-050 2-1557-050 2-1553-050 Request at [email protected]. Available as 50 µg and 100 µg

15 2. Products

2.4 TWIN-/STREP-TAG®-PROTEIN IMMOBILIZATION

Immobilization of proteins can be useful for the following applications: Antibody or serum screening Diagnostic assays Expression cloning Protein interaction studies Screening of engineered enzymes Drug screening

Via Strep-Tactin XT Antibody-free option for immobilization of Strep-tag® proteins Strep coated microplates and 8 well The ready-to-use Strep-Tactin XT coated microplates provide strips the power of the Strep-tag® system in a solid-phase, multi-well format for convenient assays and high-throughput screenings of biomolecules tagged with Twin-Strep-tag®. The combination of ® Strep-Tactin XT with the Twin-Strep-tag is highly stable with a T1/2 of 13 hours and an affinity in pM range. The strips of 8 wells are supplied in sets of 12 resulting in a 96-well plate. These 96-well plate configuration is compatible with standard multichannel pipettes, automated plate washers Related IBA products and plate readers. The biomolecules are presented to interacting Strep-Tactin XT coated microplates partners in a uniform manner which results in reliable and repro- 2-4101-001 ducible assay formats. In addition a beneficial feature of Strep-Tactin XT coated microplates is the possibility of regeneration of the plate.

Features: Oriented binding of recombinant proteins with N-terminal or C-terminal Twin-Strep-tag® Minimal non-specific binding Minimal coefficients of variation High affinity and stability of Strep-Tactin XT with Twin-Strep-tag® Cost-effective Reversibility along with high affinity

Capture of functional target proteins on Strep-Tactin XT coated microplates

Using the MTP assay the binding capacity of Strep-Tactin XT was examined and compared to Strep-Tactin. For this chromogenic color substrate purpose BAP was fused with Strep- AP reaction tag®II and applied in different amounts onto the Strep-Tactin/Strep-Tactin XT coated microplates. After washing the remaining amounts of BAP were measured. Resulting in almost 100 % recovery* of BAP-Strep-tag®II on BAP-Strep-tag®II Strep-Tactin XT compared to 8 % recovery on Strep-Tactin.

*red area

16 2. Products

Via StrepMAB-Immo StrepMAB-Immo antibody StrepMAB-Immo antibody is the reagent of choice for efficient immobilization of Strep-tag®II proteins on solid phases. StrepMAB- Immo is a murine, high-affinityStrep -tag®II specific monoclonal antibody which is especially suited for stable, mild and oriented immobilization of Strep-tag®II fusion proteins. To realize this, the antibody can be coated on e.g. microplates, columns, Biacore

CM5 sensor chips or other biochips. The nearly irreversible bind- StrepMAB-Immo, high-affinity ing is achieved for fusion proteins carrying a C- or N- terminal Strep-tag®II (Twin-Strep-tag®) specific Strep-tag®. But the Strep-tag® must be extended at the monoclonal antibody for capturing N-terminus by a SerAla linker (recombinant protein- Strep-tag® fusion proteins on solid SA-WSHPQFEK or SA-WSHPQFEK-recombinant protein). phases

StrepMAB-Immo coated microplates This antibody-based highly efficient immobilization ofStrep -tag® proteins achieves a high wash stability. StrepMAB-Immo coated microplates can be used for efficient, mild and oriented immobilization of SerAla-Strep-tag®II fusion proteins for ELISA or other assays used for protein analysis. Also small amounts of such proteins are bound with high efficiency to the microplate and will not elute during the assay due to nearly irreversible binding activity of StrepMAB-Immo.

Features: Related IBA products Ready-to-use 96-well plates for your own protein assay StrepMAB-Immo high affinity Efficient immobilization of minute amounts of antibody ® SerAla-Strep-tag II fusion proteins saves your starting material 2-1517-001 High wash stability during the assay StrepMAB-Immo coated microplates 2-1521-001

Comprehensive IBA portfolio for protein purification and analysis – product selection guide

Strep-Tactin® Strep-Tactin XT

Application and Conditions Strep-tag®II Twin-Strep-tag® Strep-tag®II Twin-Strep-tag®

Concentrated (cytosolic) 4 4 4 4

Diluted (secretion) 4 4 4 4 Purification Batch (magnetic beads) 8 4 4 4

Denatured (6M urea) 8 8 4 4

Detection (Western, ELISA) 4 4 4 4

Assay/immobil. (MTP, chips) 8 4 4 4

17 2. Products

2.5 STREP-/ HIS-TAG PRODUCTS FOR PROTEIN PURIFICATION

Purification of highly pure full-length proteins IBA provides two different Protein expression is a complex topic with many variables. There- Ni-NTA supports: fore, it is e.g. hard to predict whether a recombinant protein is ex- • Ni-NTA Sepharose for purification of 6xHistidine- tag pressed soluble or forms inclusion bodies or is partially degraded. proteins by gravity flow. To be prepared for the most common difficulties the attachment Cat.No. 2-3202-001 of two different tags at each terminus of the recombinant protein • Ni-NTA Superflow for purification provides the flexibility to obtain a highly pure and homogenous of 6xHistidine-tag proteins by protein preparation. gravity flow and FPLC

Cat.No. 2-3207-001 Important reasons for two different affinity tags on one protein are Purification of 100% full length proteins Highest purification grades Using denaturing or physiological purification conditions A smart double-tag pair is the combination of Strep-tag® and 6xHistidine-tag. Generally, it is recommended to attach one tag to the N-terminus and the other to the C-terminus.

Strep/6xHistidine-tag Starter Kit with Strep-Tactin® Superflow® high capacity This unique Starter Kit contains all reagents essential for the native purification of a double-tag protein withStrep -tag® and 6xHisti- dine-tag. The first purification is performed on a Ni-NTA Superflow® cartridge while the second purification uses a Strep-Tactin® Superflow® high capacity cartridge, selecting for 6xHistidine-tag and Strep-tag®, respectively. Cat.no. 2-1117-000

The double-tag protein purification process

Ni-NTA cartridge Strep-Tactin® Ni-NTA

Possible variants in case of proteolysis

Strep-tag®

Protein of interest Strep-Tactin® cartridge

6xHistidine -tag

Ni-NTA Resins 6xHistidine-tag is an optimal partner for Strep-tag® in double tag proteins. The combination enables the rapid control of full length expression and increases protein purity due to two independent purification methods. IBA provides all necessary products needed for the double tag purification.

18 2. Products

Gravity flowStrep MAB-Classic MacroPrep® Column An antibody-based option for Strep-tag® protein purification This antibody-based purification column withStrep MAB-Classic immobilized to MacroPrep® provides an alternative for purification of Strep-tag® proteins. Using this column as second purification step after Strep-Tactin® can increase the purity to > 95 % while only one tag – the Strep-tag®II – is used. In protein:protein interaction analysis, e.g. this column is used in the so-called One-TAP system enabling a tandem affinity purifica- Strep /6xHistidine Starter Kit tion (TAP) with two different immobilized receptors and one tag (Strep-tag®II) only. Cat.no. 2-1526-001 (1 ml); 2-1526-505 (5 x 0.2 ml)

His-STREPPER His-STREPPER The His-STREPPER adapter molecule converts a 6xHis-tag fusion protein to a Strep-tagged protein without cloning. Thereby it enables purification of initially His-tagged proteins with the Strep-tag®II:Strep-Tactin system to improve purity of the His-tag fusion protein. Cat.no. 2-0920-005 6xHis-tag Strep-Tactin fusion protein

6x His-tag/ His-STREPPER/ Fig.: Use of His-STREPPER improves Ni-NTA Strep-Tactin purity of 6xHis-tag fusion protein from 30% to 80% A 6xHis-tag fusion protein from crude bacterial cell extract was applied to Ni-NTA resin. After washing, the bound protein was eluted using Ni-NTA Elu- tion Buffer (Qiagen, lane 2). Protein purity of the 6xHis-tag fusion protein (arrow) after elution was 30% only. To improve the purity of the eluted protein a further purification step was necessary. Therefore the elution fraction was dialysed to PBS pH 8.0 and the His- STREPPER adapter molecule was added to convert the His-tag into a Target Strep-tag® fusion protein. The elution protein fraction was applied to Strep-Tactin® and the converted protein was eluted with PBS pH 8.0; 2.5 mM desthiobiotin (lane 3). The addition of His-STREPPER and purification viaStrep -Tactin® leads in this protein sample to an increase of the protein purity from 30% to 80 %. (Protein purification analysis was performed with an Agilent Bioanalyzer 2100 system instead of SDS-PAGE.).

19 10 reasons to buy immobilization, assayandinteraction The completesystemforprotein expression, purification,detection, 10. 9. 8. 7. 6. 5. 4. 3. 2. 1.

Universal DetectionSystemforWestern Blot,ELISA, No tagremoval Efficient immobilization Preserves protein complexintegrity-mildelution Economic: Low background Variable buffer Fast one-steppurification requiring alowwashingvolume Functional Highly Immunofluorescence, FACS andmore… influence protein foldingandfunction binding) or conditions, lowwashingvolumes regeneration is interaction andcompetitiveelutionwithdesthiobiotin metal ions,chelatorsorreducing agentscanbeused only -dueto pure proteins duetophysiologicalconditions re-usable Strep proteins (>95%) low non-specificbinding color controlled conditions;highsalts,detergents, MAB-Immo (non-reversible) required duetoneutral pI.Itdoesnot –specific , robust purification resins viaStrep-Tactin XT(reversible Strep

-tag Strep ® / Strep -Tactin

-tag

Version PI84-01-0001