Strep•Tag® II/Strep•Tactin® Affinity Purification Products Frequently Asked Questions

What is the principle of Strep•Tag® system?

The Strep•Tag® II purification system is based on the highly selective and easily controlled interaction between the Strep•Tag® II peptide and Strep•Tactin® resin, a specifically engineered immobilized streptavidin. The tagged protein binds to the Strep•Tactin® resin during affinity purification. Physiological buffers, like PBS, in combination with a wide range of additives can be used. After a brief wash, purified, active recombinant protein is gently eluted by adding 2.5 mM desthiobiotin to the same buffer. Desthiobiotin is an inexpensive, reversibly binding, and stable analog of biotin—the natural ligand of streptavidin. This competitive elution is the second step conferring specificity, which yields unparalleled, one-step purification. Column regeneration is visualized by a color change on the purification column.

What is the size of the Strep•Tag® II peptide and where can it be placed on the fusion protein?

The Strep•Tag® II peptide is eight amino acids (TrpSerHisProGlnPheGluLys) with a molecular weight of 1 kDa. It can be attached to the N- or the C-terminus, or between two protein domains as a linker.

What degree of purity can be expected?

Over 95%. However, impurities resulting from non-specific interactions with the recombinant protein itself could lead to lower purity. To reduce contaminants covalently linked to the recombinant protein by disulfide bonds, add reducing agents to all buffers for cell lysis and chromatography. For non-covalently linked contaminants, increase the ionic strength in all buffers for cell lysis and chromatography by adding NaCl or mild detergents (e.g., Triton® X-100, Tween® 20 detergents or CHAPS.)

Does the Strep•Tag® II peptide bind avidin?

No. Therefore, avidin can be used to block naturally occurring biotin in cell lysates and from culture medium.

For proteins expressed in the cytoplasm, is the presence of biotinylated proteins in the host organism a problem?

No. Generally, the amount of biotinylated proteins in the cytoplasm is very low and does not lead to significant inactivation of the column. In an E. coli extract derived from a 1 L culture with OD550 = 1, the total biotin content is only around 1 nmol; column capacity is 350 nmol/mL. Even the biotinylated E. coli biotin carboxyl carrier protein (BCCP) has a relatively low intracellular concentration and usually does not interfere with purification. However, to avoid binding biotin irreversibly to Strep•Tactin® resin, add avidin to the cell lysate before chromatography (20 µg/L for an E. coli culture at OD600 = 1).

For secreted proteins, is the presence of free biotin in the medium a problem (eukaryotic expression)?

Yes, it can be. The amount of biotin present depends on the cell line and the medium. Some media for insect cells or mammalian cells contain up to 800 nmol biotin per liter that has to be removed or blocked before applying the lysate to Strep•Tactin® columns. For mammalian cell

culture only DMEM and Leibovitz’s L-15 media are free of biotin. For insect cell culture, only Schneider’s medium is free of biotin. Ingredients of proprietary formulations for serum free growth are usually not disclosed, but information on biotin content can be obtained, and should be requested, from the manufacturer.

When the protein elutes from the column, is it complexed with desthiobiotin?

No. Desthiobiotin does not complex or interfere with the protein or general protein assays, and can be removed by gel filtration or dialysis

Is it possible to detect protein-protein interactions using the Strep•Tag® II system?

Yes. The One•STrEP™ Protein Interaction Kit was specifically designed to isolate intact protein complexes.

Can the Strep•Tag® II peptide be removed?

Yes. Please see vector information on page 4 to find vectors encoding enterokinase (Ek) or HRV 3C cleavage sites. However, due to the small size and chemically inert nature of Strep•Tag® II peptide, it generally does not interfere with the folding or bioactivity of the recombinant protein and does not need to be removed.

Can detergents or other buffer systems be used?

Yes. As long as the pH remains above pH 7.0, high salt, reducing reagents, chelating reagents, and detergents can be used with Strep•Tactin® resin. The resin is also compatible with BugBuster® Protein Extraction Reagent and high protein yields have been achieved using BugBuster® Master Mix to prepare cell lysates. The following table lists reagents that have been successfully tested.

 

Reagent Concentration
Reduction Agents  
DTT 50 mM
2-mercaptoethanol 50 mM
Non-ionic Detergents  
C8E4; Octyltetraoxyethylene 0.88 %
C10E5; Decylpentaoxyethylene 0.12 %
C12E8; Octaethyleneglycol Mono-n-dodecyl Ether 0.005 %
C12E9; Dodecyl nonaoxyethylene (Thesit) 0.023 %
Decyl-β-D-maltoside 0.35 %
N-dodecyl-β-D-maltoside 0.007 %
N-nonyl-β-D-glucopyranoside 0.2 %
N-octyl-β-D-glucopyranoside 2.34 %
Triton X-100 2 %
Tween 20 2 %
Ionic Detergents  
N-lauryl-sarcosine 2 %
8-HESO; N-octyl-2-hydroxy-ethylsulfoxide 1 %
SDS; Sodium-N-dodecyl sulfate 0.1 %
Zwitterionic Detergents  
CHAPS 0.1 %
DDAO; N-decyl-N,N-dimethylamine-N-oxide 0.034 %
LDAO; N-dodecyl-N,N-dimethylamine-N-oxide 0.13 %
Others  
Ammonium sulfate (NH4)2SO4 2 M
CaCl2 1 M
EDTA 50 mM
Ethanol 10 %
Guanidine 1 M
Glycerol 25 %
Imidazole 250 mM
MgCl2 1 M
NaCl 5 M
Urea 1 M

Note: These reagents have been successfully tested for purification of GAPDHStrep•Tag® II protein with concentrations up to those mentioned. Because binding depends on the sterical accessibility of the Strep•Tag® II peptide in the context of the particular protein, concentrations may deviate from the given value for different proteins.

How, and how many times, can the Strep•Tactin® resin be regenerated?

The matrix is regenerated with an azo dye, hydroxyl-azophenyl-benzoic acid (HABA), which, when applied in excess, displaces desthiobiotin. The dye is yellow in solution and shifts to red when bound by Strep•Tactin® resin, allowing visual control of the regeneration process and the functional status of the column. As long as a color gradient between the top and the bottom of the column is visible, it is not fully regenerated. The resin can be regenerated 3-5 times.

Materials

Product No. Description
71592 Strep•Tactin® Superflow™ Agarose
71608 Strep•Tactin® SpinPrep™ Kit
71613 Strep•Tactin® Buffer Kit
71603 10X Strep•Tactin® Wash Buffer
71553 pET-51b(+) DNA
71570 pET-51 Ek/LIC Vector Kit
71554 pET-52b(+) DNA
71571 pET-52 3C/LIC Vector Kit
71572 pIEx-8 Ek/LIC Vector Kit
71557 pIEx-10 DNA - Novagen
71574 pIEx-10 Ek/LIC Vector Kit
71558 pTriEx™-5 DNA - Novagen
71575 pTriEx-5 Ek/LIC Vector Kit
71559-M pTriEx™-6 DNA - Novagen
71576 pTriEx-6 3C/LIC Vector Kit
71577 pTriEx-7 Ek/LIC Vector Kit
70584-M BugBuster® Protein Extraction Reagent
71456 BugBuster® Master Mix