Frequently asked questions Strep-tag®
Do you have any questions about our Strep-tag® technology or do you want to inform yourself in the trouble shooting guide? Then browse through our FAQs and troubleshooting guides.
In our FAQs we discuss frequently occurring problems and provide solutions. Trust IBA's many years of experience with the proprietary Strep-tag® technology and get useful advice on how to improve your research results.
The highly selective Strep-tag® system is based on one of the strongest non-covalent interactions in nature, which is the interaction of biotin to streptavidin. The easily and straight-forward Strep-tag® technology allows the purification, detection and immobilization of recombinant proteins.
During affinity purification the Strep-tag® fusion protein of interest binds to an immobilized supporting material/matrix (e.g. agarose bead) which is coated with a specifically engineered streptavidin variant named Strep-Tactin® or Strep- Tactin®XT.
The system includes two affinity tags.
- Strep-tag®II - a synthetic peptide consisting of eight amino acids (WSHPQFEK).
- Twin-Strep-tag® which consists of two Strep-tag®II sequences in series with an internal linker region ((WSHPQFEK (GGGS)2 GGSAWSHPQFEK), with GS-linker; total size of 28 amino acids).
The tag strongly depends on your needs in your final experimental approach. Due to its small size and chemically inert nature, Strep-tag®II does generally not interfere with the folding or bioactivity of the recombinant protein.
The Twin-Strep-tag® enables the same mild and rapid purification as Strep-tag®II but, in addition, has an increased affinity for Strep-Tactin® and Strep-Tactin®XT which allows efficient purification (even in batch or directly from cell culture supernatants). With the Twin-Strep-tag® we introduce an avidity effect, which consequently reduces the off-rate of the total-tag and finally enhances the binding affinity to Strep-Tactin® as well as Strep-Tactin ®XT. After binding of the protein and changing to wash buffer the curve stays on a high level since a Twin-Strep-tag® fusion protein dissociates completely, only when both tags leave their binding pockets at the same time. If one tag dissociates it is kept in close proximity to its pocket by the other tag and is likely to re-bind. Therefore the dynamic equilibrium is on the side of the bound protein complex.
Both tags can be N- or C- terminally fused to recombinant proteins.
We recommend to choose two small, neutral amino acids (like S, A or G). Please try to avoid big, aromatic, charged or structurally potent residues. In our vectors, linkers are already included.
The best support for the purification depends on the recombinant protein and is normally not predictable. However, Strep-Tactin®XT is the further engineered variant of Strep-Tactin® and has a binding affinity in the nM range to Strep-tag®II and to the Twin-Strep-tag® in the low pM range. The high binding affinity of Strep-Tactin®XT ensures higher protein yields as well as higher purity compared to Strep-Tactin®.
Strep-Tactin®XT requires elution of the target protein with Buffer BXT containing 50 mM Biotin. Due to the high binding affinity desthiobiotin cannot be used for elution. For Regeneration of the Strep-Tactin®XT, we recommend the application of Buffer XT-R (3 M MgCl2). Otherwise, freshly prepared 10 mM NaOH ca be used as well. Since biotin is used for elution of the target protein, Buffer R with HABA is not applicable for Strep-Tactin®XT. However, Buffer R with HABA can be used to monitor a successful regeneration of the resin subsequent to Buffer XT-R treatment.
In case of optimal handling and storage the columns can be re-used 3-5 times.
You can easily check this with HABA. HABA has a yellow color in solution. It binds to the biotin binding pocket of Strep-Tactin®, thereby its color changes to red in case of Strep-Tactin® (see left side) or to orange in case of Strep-Tactin®XT (see right side). As long as this color shift happens you can re-use the column. HABA can be easily washed off the column with wash buffer
Yes, we offer a Strep-Tactin®XT 4Flow® Starter Kit [Cat No: 2-5998-000]. The kit comes with a 1 ml Strep-Tactin®XT 4Flow® column and all necessary buffers for purification (buffer W, buffer XT-R and buffer BXT) and a detection reagent for western blot.
In case of a 1 ml column please do the following:
Take your column and close the lower cap. Sway the resin (50 % suspension) until a homogenous suspension is obtained. Then fill the column with 2 ml resin (which corresponds to 1 ml bed volume). Note: Please use per 1 liter cell extract about 2 ml resin. Add 2 CVs (column bed volumes) of buffer W and resuspend your resin by stirring with a small spatula (to avoid air bubbles, which could reduce your flow rate). Then let the resin settle down for 10 to 15 minutes. Carefully place the soaked upper frit on top of the column bed by slowly pushing the frit with the back of a Pasteur pipet. Please take care not to compress the resin. Finally wash your resin with 2 CVs of buffer W. Optionally: Check upper frit position and if necessary re-adjust it.
The material will be harmed and will not tolerate -20°C.
Strep-Tactin®: pH 7.5 – 8
Strep-Tactin®XT Superflow®: pH 6 – 10
Strep-Tactin®XT 4Flow®: pH 4 – 10
Physiological buffers (e.g. PBS) in combination with a wide range of additives (high salt, detergents, reducing agents, metal ions and chelating agents) can be used. The complete list of tested additives for Strep-Tactin® or Strep-Tactin®XT can be found in die pdfs below.
Our buffers contain EDTA because it is an antimicrobial agent. The buffers will also work without it and you can prepare the buffers your own without EDTA.
Please note that preparing a 50 mM biotin solution is a time-consuming procedure which takes several hours. Therefore we recommend to buy our ready-to-use buffer BXT (#2-1042-025) if possible!
If you have to prepare the biotin solution your own you have to consider the following:
Use buffer W for preparing buffer BXT. Biotin does not dissolve under acidic conditions it requires alkaline conditions. In addition biotin decreases the pH of the buffer during the solvation process.
Therefore the pH needs to be measured continuously during the solvation procedure and has to be adjusted from time to time with sodium hydroxide to increase the pH and to get the biotin stepwise solved.
Contamination of biotin is often a problem during mammalian expression, when the protein is secreted into the medium. Mammalian expression media contain high amounts of biotin (vitamin H). Biotin will block the column and the Strep-tag®II or Twin-Strep-tag® target protein cannot bind to the resin. Therefore it needs to be blocked before it can be applied to a Strep-Tactin® or Strep-Tactin®XT column. For this the BioLock solution (Biotin blocking solution) can be used. Per 70 µg of biotin add 1 ml of this solution. Further information can be found here.
Intrinsic biotin or biotinylated proteins are only abundant in very low concentrations and do not disturb the purification process or the purity of the target protein. To completely avoid those proteins small amounts of Avidin or BioLock can be added.
In general the interaction of Strep-tag®II and Twin-Strep-tag® to Strep-Tactin®XT is quite stable and not influenced by detergents or other additives. A list of so far tested reagents can be found here.