Both can be easily checked with HABA (Buffer R). HABA has a yellow color in solution and binds to the biotin binding pocket of Strep-Tactin® and Strep-Tactin®XT, respectively. As a consequence, it changes color to red in case of Strep-Tactin® or to orange in case of Strep-Tactin®XT. As long as this color shift is observed, the column can be re-used. Subsequently, HABA can be simply washed off with Buffer W.

Watch our tutorials on color based functionality check here:

Strep-Tactin®XT 4Flow®

To get high protein concentrations in one fraction, add 0.6 CV of Buffer BXT as elution fraction 1 (E1), then 1.6 CV (E2), and finally 0.8 CV (E3). Main protein content should be in E2. 

Biotin is a common ingredient in cell culture media. Free biotin binds to Strep-Tactin® and consequently inactivates the resins. It has to be removed or masked prior to affinity chromatography. For this purpose, we recommend our ready-to use Biotin Blocking Solution (BioLock) or avidin. The required amount of blocking solution can be checked here.

Please have a look at our compatible reagents list for Strep-Tactin®XT.

Please have a look at our compatible reagents list for Strep-Tactin®.

Yes. Please note that in order to release the biotinylated proteins from the resin, biotin has to be added. As it irreversibly binds to the resin, this cannot be regenerated. Desthiobiotin is not applicable, because it does not displace biotinylated proteins from the resin.

Strep-tag®II and Twin-Strep-tag® do generally not interfere with the folding or bioactivity of the recombinant protein. However, if the tag has to be removed, we recommend the application of the TEV protease.

No, desthiobiotin does not bind to the target protein. Desthiobiotin can be removed from eluate via gel filtration or dialysis.

No, biotin does not bind to the target protein. Biotin can be removed from eluate via gel filtration or dialysis.

Packing of a gravity flow column video tutorial

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). Add 2 column bed volumes (CV) 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 at least 30 minutes. Soak top frit in 1x Buffer W.
  
• 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 to not compress the resin. Finally wash your resin with 2 CV of Buffer W.
• Optionally: Check upper frit position and if necessary, re-adjust it.

Our buffers contain EDTA because it is an antimicrobial agent. The buffers will also work without it and you can prepare the buffers on your own without EDTA.

Biotin can be removed by gel filtration or dialysis.

Desthiobiotin can be removed by gel filtration or dialysis.

When the column is taken from the cold storage room to the bench, the increased temperatures can cause small air bubbles in the column. The reason is that buffers are able to take up more gas at lower temperatures than at room temperature. To avoid bubbles, keep on working in the cold room, use degased buffers or wash the column with buffers equilibrated at room temperature immediately after it is transferred to the bench.

Free Biotin inactivates Strep-Tactin® resins and has to be removed or masked prior to affinity chromatography. The simplest way to get rid of biotin prior to purification is the addition of the avidin-containing biotin blocking solution BioLock. Avidin binds to biotin as well as biotinylated proteins and thus prevents their binding to the Strep-Tactin® resin.

The multimer of Strep-Tactin® as well as Strep-Tactin®XT has a molecular weight of 52 kDa. Both are tetramers and each subunit has a molecular weight of 13 kDa.

It is normal that the flow speed varies between columns. If the lysate is not clear enough or to viscous, the column can clog. You could try to centrifuge the supernatant at a higher speed (18,000 x g, 5 minutes, 4 °C) and to filtrate the lysate with syringe filters (pore size: 0.2 - 0.45 µm) directly before the application onto the column. If the lysate is very viscous, the application of RNase A and DNase I is recommended.

To apply large sample volumes onto a gravity flow column with continuous flow, the WET FRED applicator is recommended. This device works 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 do not need supervision. Furthermore, no sophisticated software is necessary, facilitating set up and use.

See how the Wet Fred is set up and cleaned after use in the video tutorials.

1. The pH might be incorrect. The pH should be between 4 and 10 for Strep-Tactin®XT 4Flow® resin. 
2. The Strep-tag®II or Twin-Strep-tag® are not present or have been cleaved: use protease deficient E. coli expression strains or add protease inhibitors during cell lysis to avoid tag degradation; check that the tag is not associated with a portion of the protein that is processed. 
3. If Strep-tag®II or Twin-Strep-tag® are not accessible, fuse the tag to the other protein terminus or use a different linker. If the Strep-tag®II is used, replace it by the Twin-Strep-tag®. The larger size of the Twin-Strep-tag® can enhance the accessibility.

StrepMAB-Immo is not recommended for western blot, but is perfectly suitable for capturing Strep-tag® proteins on solid surfaces (e.g. SPR, microtiter plates). It is important to know that the StrepMAB-Immo antibody requires a SA-linker in order to bind with highest specificity and affinity. For western blot, please use StrepMAB-Classic HRP or Strep-Tactin® HRP.

For 1000 ml 1x Buffer BXT, take 800 ml 1x Buffer W (Wash buffer: 100 mM Tris/HCl pH 8.0, 150 mM NaCl, 1 mM EDTA), and add 12.2 g biotin. Biotin only dissolves under alkaline conditions, but during the solvation process, the pH value of the buffer decreases. Therefore, the pH value needs to be continuously measured and occasionally adjusted to approximately pH 8 with sodium hydroxide. After biotin is completely solved, the solution is filled up to 1000 ml with 1x Buffer W.

Under ideal conditions, the resin can be re-used >10 times without loss in performance.

20 ml suspension corresponds to 10 ml resin. But in 1 ml column, there is 1 ml resin.

Both ligands (Strep-Tactin® and Strep-Tactin®XT) can be combined with both tags (Strep-tag®II and Twin-Strep-tag®). However, Strep-Tactin®XT shows a binding affinity in the nM range when combined with the Strep-tag®II and in the pM range with the Twin-Strep-tag®, whereas Strep-Tactin® has a binding affinity in the µM range for the Strep-tag®II and in the nM range for the Twin-Strep-tag®. The high binding affinity of Strep-Tactin®XT ensures higher protein yields as well as higher purity compared to Strep-Tactin®. Furthermore, the combination of Strep-Tactin®XT and Twin-Strep-tag® is more suitable for analytical applications.

Strep-Tactin® 4Flow®: pH 7 to 8
Strep-Tactin®XT 4Flow®: pH 4 to 10
MagStrep® Strep-Tactin®XT beads: pH 6 to 10

We recommend to choose two small, neutral amino acids, like serine-alanine. Please try to avoid big, aromatic, charged or structurally potent residues. In our vectors, linkers are already included.

Strep-tag® II has eight amino acids (WSHPQFEK) and a molecular weight of 1058 Da.

Yes, it is also possible to regenerate Strep-Tactin® 4Flow® and Strep-Tactin®XT 4Flow® resins with 100 mM NaOH. Add 15 column volumes (CV) 100 mM NaOH. Directly afterwards, add 15 CV Buffer W. Please do not incubate with NaOH for a longer period. We recommend the use of NaOH for cleaning the column from unspecifically bound or precipitated protein.

The binding affinity is in the micromolar range.

The binding affinity is in the nanomolar range.

The binding affinity is in the nanomolar range.

The binding affinity is in the picomolar range.

The binding affinity is in the micromolar range.

The binding affinity is in the picomolar range.

The antibody is reacting with the MW marker. Add a blank lane between the MW marker and the first sample lane.

For blocking biotinylated proteins on western blots, the application of Biotin Blocking Buffer containing avidin is recommended.

Strep-Tactin®-AP (2-1503-001), Strep-Tactin®-HRP (2-1502-001) or StrepMAB-Classic HRP (2-1509-001) can be used to detect Strep-tag®II and Twin-Strep-tag® proteins in western blots. 

No, skimmed milk is not applicable, since it contains biotin which would be detected by Strep-Tactin® HRP, and thus produces high background. Please use BSA.

No, skimmed milk is not applicable, since it contains biotin which would be detected by Strep-Tactin® AP, and thus produces high background. Please use BSA.

Yes, you can use PVDF instead of nitrocellulose. Please keep in mind that PVDF leads to higher background compared to nitrocellulose.

1. Please always include a positive and a negative control to check if the antibody or Strep-Tactin® conjugate is functional.
2. The primary antibody and the secondary antibody are not compatible. Use a secondary antibody that was raised against the species, in which the primary was raised (e.g. primary is raised in rabbit, use anti-rabbit secondary). Alternatively, conjugated Strep-Tactin can be used for Strep-tagged proteins avoiding incompatible antibody pairs. 
3. Not enough primary or secondary antibody is bound to the protein of interest. Use higher antibody concentrations or incubate longer, e.g. overnight at 4 °C. 
4. Cross-reaction between blocking agent and primary or secondary antibody. Use a mild detergent such as Tween 20 or switch blocking reagent. Commonly used blocking reagents are e.g. milk, BSA, serum or gelatin. Please keep in mind that milk cannot be used for blocking if the detection should occur with Strep-Tactin® conjugates, since it contains biotin which is detected by Strep-Tactin®, and therefore produces high background. 
5. The amount of antigen could be insufficient. Use protease inhibitors during the preparation, and load at least an aggregate of 20-30 μg protein per lane. 
6. The protein of interest is not abundantly present in the tissue. Use an enrichment step to maximize the signal, e.g. prepare nuclear lysates for a nuclear protein. 
7. The transfer of the protein to the membrane was poor. Check the transfer with a reversible stain such as Ponceau S. Make sure that the transfer was not performed the wrong way. If using PVDF, please keep in mind that the membrane needs to be activated with methanol prior to blotting. 
8. The membrane was washed too excessively. Reduce the quantity of wasing steps or the time per step. 
9. Too much blocking does not allow you to visualize your protein of interest. Switch blocking reagents or block for less time, we recommend 3% BSA and 0.05 % v/v Tween 20 in PBS for 60 min. 
10. The antibody is over-used. Use fresh antibody as the effective concentration is lowered upon each re-use. 
11. The Antibody or Strep-Tactin® conjugate is inhibited by sodium azide. Do not use sodium azide together with HRP-conjugated antibodies. 
12. The detection kit is too old and substrate is inactive. Use fresh substrate.

1. Blocking of non-specific binding might be absent or insufficient. Increase the blocking incubation period and consider changing blocking agent. We recommend 3% BSA and 0.05% v/v Tween 20 in PBS for 60 min. These can be included in the antibody buffers as well.
2. The antibody or Strep-Tactin® conjugate concentration could be too high. Titrate the antibody to the optimal concentration and incubate for a longer period with the higher dilution.  
3. Incubation temperature may be too high. Incubate blot at 4 °C. 
4. The secondary antibody may be binding non-specifically or reacting with the blocking reagent. Run a secondary antibody control without primary antibody. 
5. Cross-reaction between blocking agent and primary or secondary antibody. Add a mild detergent such as Tween 20 to the incubation and washing buffer. 
6. Washing of unbound antibodies may be insufficient. Increase the number of washes. 
7. Your choice of membrane may give high background. Nitrocellulose membranes are considered to cause less background than PVDF. 
8. The membrane has dried out. Care should be taken to prevent the membrane from drying out during incubation.

1. Cell lines that have been frequently passaged gradually accumulate differences in their protein expression profiles. Return to the original non-passaged cell line and run the current and original cell line samples in parallel.
2. The protein sample has multiple modified forms in vivo such as acetylation, methylation, myristylation, phosphorylation, glycosylation etc. Examine the literature and use an agent to dephosphorylate, de-glycosylate, etc. the protein to bring it to the correct size. 
3. The target in your protein sample has been digested (more likely if the bands are of lower molecular weight). Make sure that you incorporate sufficient protease inhibitors in your sample buffer. 
4. The antibody or Strep-Tactin® conjugate concentration is too high. At high concentrations, multiple bands are often detectable. Try decreasing the antibody concentration and/or the incubation period. 
5. The target protein may form multimers. Try boiling in SDS-PAGE sample buffer for 10 minutes rather than 5 minutes to disrupt multimers.

Air bubbles were trapped against the membrane during transfer or the antibody is not evenly spread on the membrane. Make sure you remove bubbles when preparing the gel for transfer. Incubate antibodies under agitation.

The antibodies are binding to the blocking agent. Filter the blocking agent. 

Too much antibody or Strep-Tactin® conjugate. Use a higher dilution of the antibody or Strep-Tactin® conjugate.

Yes, they can be dissolved in DMSO.

We recommend storing peptides in aliquots to avoid multiple freeze-thaw cycles.