IBA Product FAQs

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®

Strep-Tactin® Sepharose®

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 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®.

MagStrep "type3" XT beads are coated with Strep-Tactin®XT. The affinity of Strep-Tactin®XT to biotinylated proteins is too low, leading to inefficient immobilization. For efficient immobilization, it is recommendable to use Strep-Tactin® resins. 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.

No, the affinity of Strep-Tactin®XT to biotin is too low. Please use one of the Strep-Tactin® resins. 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.

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.

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.

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 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 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.

Packing of a gravity flow column video tutorial

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 6 and 10 for Strep-Tactin®XT Superflow resins and between 4 and 10 for Strep-Tactin®XT 4Flow® resin, respectively. 
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.

1. The pH might be incorrect. The pH should between 7 and 8. 
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.

There are several Strep-Tactin® resin versions available. All resins differ in their properties and suitability for applications. Learn more about the different resin matrices in order to know when to use which resin.

Click here to see the specifications for Strep-Tactin® purification resins.

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.

The resin can be re-used 3 - 5 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.

HABA binds to the same Strep-Tactin® binding pocket as Strep-tag® and desthiobiotin (or biotin), but only with very low affinity. Nevertheless, it accelerates desthiobiotin removal if applied in excess. That means each time when a Strep-Tactin®:desthiobiotin complex dissociates on the column, the resulting free Strep-Tactin® binding site may be occupied by HABA or desthiobiotin in competition. As HABA is applied in excess, desthiobiotin has only a reduced chance to find a free biotin binding pocket, which finally results in accelerated removal of desthiobiotin from the column.

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 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.

All Strep-Tactin® resins: pH 7.5 to 8

Strep-Tactin®XT Superflow®: pH 6 to 10

Strep-Tactin®XT 4Flow®: pH 4 to 10

The highly selective Strep-tag® system is based on one of the strongest non-covalent interactions in nature, which is the interaction of biotin and streptavidin. The easy 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. 

Yes, it is also possible to regenerate the columns with 0.5 M NaOH. Add 5 column volumes (CV) 0.5 M 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.

For removal of desthiobiotin, please use Buffer R with HABA. Buffer R is the specific and smooth cleaning buffer and will not damage the Strep-Tactin® resin, whereas the longer use of NaOH will affect the resin in the long term, causing the loss of binding capacity.

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

Twin-Strep-tag® has 28 amino acids (WSHPQFEK-GGGSGGGSGG-SA-WSHPQFEK) and a molecular weight of 2887 Da.

Both tags can be attached to the N- or the C-terminus, or between two protein domains as a linker.

We recommend to choose two small, neutral amino acids (like S, A or G). Please try to avoid large, aromatic, charged or structurally potent residues. IBA´s expression vectors already include such linkers.

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.

No. Therefore, avidin can be used in detection assays in order to mask naturally occurring biotinylated proteins which would generate background signals if present in the probe.

Yes, but normally it is not necessary. Due to the small size and chemically inert nature, Strep-tag®II and Twin-Strep-tag® do generally not interfere with the folding or bioactivity of the recombinant protein.

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 nitrocellulose instead of PVDF. 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.

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. 

No, biotin does not cause a conformational change in the Fab-Streps/Nano-Streps. Due to its higher affinity, biotin displaces the Fab-Streps/Nano-Streps in the Strep-Tactin® binding pockets. This disrupts the multimerization of Fab-Streps/Nano-Streps. Due to their low affinity as monomers, they spontaneously dissociate from the cells.

Yes, this is possible. However, if a high affinity antibody is used, it might remain attached to the cell surface after monomerization. Therefore, after elution, the isolated cell population might not be label-free.

To bind inside the column, the used Fab fragments/antibodies/nanobodies should have a Twin-Strep-tag®, which efficiently binds to Strep-Tactin® to capture cells. Alternatively, it is also possible to use e.g. a desthiobiotinylated antibody. Elution with biotin is still possible here. Biotinylated antibodies bind irreversibly to the column and therefore we recommend using the antibodies only for negative cell selection or depletion approaches.

For protein isolation we recommend our resins and columns specifically developed for this application.

Fab-Streps/Nano-Streps do not stably bind to cells as monomers. Multimerization of Fab-Streps/Nano-Streps on Strep-Tactin® backbones is necessary to increase the avidity and ensure stable binding to the cell surface.

The “Fab” in a Fab-Strep is derived from a Fab fragment of a conventional antibody. It is subsequently fused to a Twin-Strep-tag® and modified to achieve a low affinity to the target. “Nano” deviates from nanobodies, which are derived from camelid heavy chain antibodies. 

Fab-Streps/Nano-Streps are fused to a Twin-Strep-tag®, which is a peptide sequence consisting out of 28 amino acids. The Twin-Strep-tag® binds to Strep-Tactin®. The addition of biotin reverses the binding.

Fab-Streps/Nano-Streps are suitable for different cell isolation approaches (fluorescent, magnetic or affinity-chromatographic cell isolation) based on our Strep-tag® technology (Fab-TACS®/Nano-TACS® - Traceless Affinity Cell Selection). For the different approaches, Fab-Streps/Nano-Streps have to be combined with either fluoresecent Strep-Tactin®, Strep-Tactin® Magnetic Microbeads or Strep-Tactin® TACS Agarose columns. Affinity-chromatographic exosome isolation (Fab-TACS® for exosomes) is also possible.

Around 50 kDa (the exact molecular weight depends on the Fab-Strep).

Around 15 kDa (the exact molecular weight depends on the Nano-Strep).

We recommend storing Fab-Strep/NanoStrep in aliquots to avoid multiple freeze-thaw cycles.

Yes, if the method is using similar paramagnetic beads in size (µm) the required magnetic fields are similar making magnets and magnetic beads interchangeable in most cases. Magnetic bead systems using nano-sized magnetic beads have a different field strength in their magnet.

Strep-Tactin® Magnetic Microbeads cannot be used in combination with the MACS system offered by competitors designed for their magnetic microbeads, as these particles are nanoparticles.

If Strep-Tactin® PE/APC was frozen, we cannot guarantee that it still works. Depending on the temperature and time it might still work. We highly recommend testing samples which were accidentally frozen before using them in an experiment. 

We do not provide a molar extinction coefficient for the conjugates, only for the components. They are as follows: APC: ε1% 650 nm = 70, PE: ε1% 566 nm = 82, Strep-Tactin® A280 nm = 26,7

Strep-Tactin® Magnetic Microbeads contain multimeric Strep-Tactin®. The multimers are heterogeneous in size consisting of different amounts of tetrameric Strep-Tactin (which has a molecular weight of 50 kDa). We do not give an exact molecular weight.

Strep-Tactin® Magnetic Microbeads cannot be used in combination with MACS system offered by competitors designed for their magnetic microbeads, as these particles are nanoparticles. 

A method for concentration is the use of e.g., Vivaspin columns (Centricon columns are also possible), but all efforts to concentrate samples can also lead to a loss of isolated exosomes. The loss can range between 25% and 30%, but is dependent on the initial concentration and the sample type. With this method however, you can combine all fractions and significantly reduce the volume and achieve concentrated exosomes. Vacuum centrifugation is not recommended. Salts from your buffer will also be concentrated, which will destabilize the membrane of the isolated exosomes.

Sepharose is used for size exclusion chromatography and this method does not select exosomes based on surface marker expression. This can result in contaminations with non-exosome particles that have the same size, e.g., protein complexes. The Fab-TACS® system for exosomes is an affinity-chromatography-based approach that specifically targets CD9 or CD81 on the surface of exosomes, which results in a high purity. The selection reagents are reversible, therefore you get unlabeled exosomes similar to what you can achieve with sepharose-based size exclusion chromatography. A disadvantage of the Fab-TACS® system is that some reagents are still present in the eluate (Fab-Streps and biotin) although they are not bound to the exosomes. However, this does not affect downstream applications such as NTA, Western Blot or RNA analysis. If needed, the compounds can be easily removed by an additional size exclusion chromatography step.

MHC I-Streps are fused to a Twin-Strep-tag®, which is a peptide sequence consisting out of 28 amino acids. The Twin-Strep-tag® binds to Strep-Tactin®. The addition of biotin reverses the binding.

For fluorescent cell staining & sorting, MHC I-Streps should be combined with fluorescent Strep-Tactin®. We currently offer Strep-Tactin® PE and Strep-Tactin® APC. See our protocol for detailed instructions.

For magnetic cell isolation, MHC I-Streps should be combined with Strep-Tactin® Magnetic Microbeads.

In principle this is possible, because MHC I-Strep can be loaded into our Strep-Tactin® TACS Agarose columns. However, we only recommend this approach if target cell numbers higher than 1 x 107 are expected. For further discussion please contact our product management.

In principle it is possible to use biotinylated MHC I complexes, because they will bind to Strep-Tactin® conjugates. Note: Biotin addition will not cause the monomerization and thereby dissociation of isolation reagents. The sorted/isolated cell population will not be label-free. If cells are just fluorescently stained and analysed, using biotinylated MHC I complexes in combination with fluorescent Strep-Tactin® is not a problem, for in vitro or in vivo studies isolation reagents can interfere with biological functionality.

MHC I-Streps are expressed in E. coli.

Depending on the MHC I, the molecular weight ranges between 45-50 kDa.

The pre-mix can be stored at 4 °C (do not freeze), but should be used within 48 h.  We highly recommend to always make a fresh mix of the components.

Yes, this is possible.

No, the T cells will further expand. It just removes the activation stimulus, generating a more defined and controllable cell signal.

Yes, they can be dissolved in DMSO.

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

The peptides are lyophilized in 0.1% TFA (Trifluoroacetic acid)/water/ACN (acetonitrile) solution. Water and ACN will be removed in the process, but TFA remains.