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Strep-tag®: The leading affinity tag in recombinant protein production

The Strep-tag® system enables cloning, expressiondetectionpurification, as well as further analysis of recombinant proteins. The highly specific interaction of the Strep-tag®II with Strep-Tactin® ensures efficient one-step purification of the protein of interest in unparalleled purity even from crude cell lysates.

The Strep-tag® system is compatible with a large number of protein classes, e.g., metalloproteins, membrane proteins and fragile protein complexes with multiple subunits. In addition, the mild and physiological conditions promote the yield of fully functional proteins, making the system particularly suitable for purification of enzymes as well as structural investigations, protein-protein interaction studies, ligand-receptor investigations or even separation of living cells for re-culturing processes. Simultaneously, a high tolerance towards different buffers and additives promotes its universal applicability.

Besides the advantages of the Strep-tag® technology for efficient protein purification, the near covalent affinity (pM range) of Twin-Strep-tag® to Strep-Tactin®XT expands the range of applications towards protein analysis e.g., ELISAsurface plasmon resonance (SPR) or bio-layer interferometry (BLI).

The Strep-tag® technology represents a universal toolbox that provides solutions for the entire recombinant protein production chain

The Strep-tag® technology represents a universal toolbox that provides solutions for the entire recombinant protein production chain: from cloning, protein expression and purification to further analysis like detection, immobilization and assay applications.

One tag - Multiple applications

Highest affinity tag for protein purification

The Strep-tag® protein purification system comprises two affinity tags, the 8AA Strep-tag®II and its tandem version Twin-Strep-tag®. Both versions can bind to Strep-Tactin® and its high affinity variant Strep-Tactin®XT. Thereby the two tags differ in the affinities with which they bind to Strep-Tactin® and Strep-Tactin®XT. Depending on the application and properties of the protein of interest one can combine the different tags and Strep-Tactin® variants according to the required affinity.

It is well known for its outstanding performance with regard to purifying recombinant proteins in a simple procedure with highest purities. Its latest development - the Strep-Tactin®XT - provides a binding affinity in low pM ranges in combination with Twin-Strep-tag® still maintaining binding reversibility and mild recovery of immobilized proteins. This improvement in affinity allows protein purification at high yields and purity, even for challenging proteins and from mammalian expression systems (e.g. Expi). Read more about the benefits of Strep-tag® purification in combination with high density mammalian protein expression in our white paper.

Furthermore, it fulfills the high demands of protein interaction analysis or assays and downstream applications like SPR, thus covering all steps from purification to immobilization efficiently.

We offer specialized matrices for any demand in form of ready to use kits, FPLC columns, gravity flow or spin columns, suspensions, coated microplates, magentic beads, and more (find products in our web shop). Additionally, we provide innovative solutions for approaches surrounding batch purification, FPLC/HPLC, high-throughput screens and assay development

Strep-tag®  protein purification system at a glance

Key features:

  • Highly selective binding properties leading to unparalleled protein purity (> 95 %)
  • Bioactive target proteins due to the rapid one-step purification under target specific conditions
  • Variability in buffer conditions, e.g. high salts, detergents, metal ions, chelators or reducing agents
  • Peptide tag with balanced/inert amino acid composition and, therefore, the protein structure or activity is not influenced, and removal of tag is not required
  • Favorable for protein:protein interaction studies due to mild elution conditions and low washing volumes
  • Universal toolbox including products for cloning, purification, and analytic applications (ELISAFACSSPR, Microscopy, Western Blot, etc.) 

When should the Strep-tag® purification system be the method of choice?

Besides the purification of classical proteins the Strep-tag® system should be the method of choice for:
  • metalloproteins
  • membrane proteins
  • low abundant proteins (in combination with Strep-Tactin®XT)
  • sensitive protein complexes with multiple subunits
  • bioactive proteins

Is There An Alternative To His-tag?

Choosing an appropriate affinity chromatography system for simple and efficient protein purification is a common question. There are differences between the systems which rarely are clearly represented, making it hard for scientist to reach a decision.

This comprehensive comparison of the most frequently used systems, His-tag and Strep-tag®, explains these differences as well as recommends one system depending on the properties of the target protein, expression host and purification conditions.

Download white paper (pdf)


Strep-tag® system - quick & easy protein purification protocol 

The Strep-tag® technology allows efficient one-step purification of Strep-tag®II or Twin-Strep-tag® proteins via affinity chromatography. The system is distinguished by quick and simple purification protocol, regardless of the target protein class.
This step-by-step tutorial demonstrates the full purification procedure of a target protein, here mCherry-Twin-Strep-tag, via gravity flow column, including the initial buffer and column preparation steps, as well as column regeneration after purification and its’ preparation for storage until further use. Gravity flow column used in the tutorial contained high-performance Strep-Tactin®XT 4Flow® resin.

Thumbnail for protein purification full how-to video

What are the differences between Strep-Tactin® and Strep-Tactin®XT? 

Which resin to choose when purifying strep-tagged proteins?

Strep-Tactin® and its high-affinity counterpart, Strep-Tactin®XT, are both streptavidin-derived mutants. Each of these proteins weighs 52 kDa, comprising four subunits, each housing a biotin binding pocket.
While streptavidin binds biotin with an almost irreversible affinity in its pocket, Strep-Tactin® and Strep-Tactin®XT exhibit reduced affinity for biotin. However, they still maintain the capability to bind biotin. The biotin binding pockets on both Strep-Tactin® and Strep-Tactin®XT have been optimized for binding with Strep-tag® and its tandem version, Twin-Strep-tag®. In contrast to the near irreversibility of biotin binding with streptavidin, the interaction with either tag on Strep-Tactin® or Strep-Tactin®XT is reversible.
Compared to streptavidin, a crucial alteration in Strep-Tactin® involves mutating the lid-like loop, resulting in a consistently open confirmation of the loop. This initial mutation enhances the affinity for strep-tagged proteins by a factor of ten. Upon peptide binding, the lid remains open, allowing for easy removal of the peptide by competitive biotin.
In Strep-Tactin®XT, a second mutation was introduced on the opposite side of the chain. This alteration enhances the interaction between the peptide and the biotin binding pocket, significantly boosting the binding affinity.
More detail about the improvements of the sequence can be found in Schmidt et al. 2021.

Streptavidin & biotin

Strep-Tactin® & Strep-tag®

Strep-Tactin®XT & Twin-Strep-tag®

Strep-Tactin® and Strep-Tactin®XT purification cycle

For the purification of Strep-tag®II or Twin-Strep-tag® proteins both resin types, Strep-Tactin® and Strep-Tactin®XT, are applicable. Protein purification with Strep-Tactin® as well as Strep-Tactin®XT is very simple and fast, since the purification cycle for each target protein is the same. However, if a target protein needs a specific buffer composition, e.g. PBS, HEPES or no chelating agents, like EDTA, the buffers can be easily adapted without the need of changing the purification cycle. Both resin types accept various buffer compositions, reagents and additives and an overview is given in the compatible reagents list for Strep-Tactin® as well as Strep-Tactin®XT.
The purification cycles for both resin types are highly similar. However, the sample application, the elution, and the regeneration step are slightly different and will be explained in the following. 

A comparison of purification via Strep-Tactin® and Strep-Tactin®XT depicts two changes in the procedure. The first and second step (lysate application & wash) remain the same. But the elution and the regeneration streps are different for both systems. For elution from Strep-Tactin® desthiobiotin is used whereas Strep-Tactin®XT requires biotin for elution. Also the regeneration step differs. HABA is used for regeneration from Strep-Tactin® and in case of Strep-Tactin®XT 3 M MgCl2 is applied.

 

 Strep-Tactin® 

 Strep-Tactin®XT

 Binding affinity

 Strep-tag® II: μM range
 Twin-Strep-tag®: nM range
 Strep-tag® II: nM range
 Twin-Strep-tag®: pM range

 Elution

 Elution with Buffer E
 (2.5 mM Desthiobiotin)
 Elution with Buffer BXT
 (50 mM Biotin)

 Regeneration

 Elution with Buffer R (HABA)  Elution with 10 mM NaOH or 3 M MgCL2
Strep-Tactin® protein purification cycle
Strep-Tactin®XT protein purification cycle

Properties of Strep-Tactin® and Strep-Tactin®XT

The shared benefits of Strep-Tactin® and Strep-Tactin®XT are

  • Purification of bioactive recombinant proteins under physiological conditions
  • Protein aggregation is avoided
  • Broad range of detergents, chelators, salt or redox conditions allowed
  • Highly specific interaction of Strep-tag® with Strep-Tactin®, no unspecific protein binding (high specificity in screening and assays)

Additional benefits of Strep-Tactin®XT due to increased binding affinity in pM range:

  • Efficient, directed, and reliable immobilization of targets from complex mixtures while maintaining the reversibility
  • Allows all kinds of analytic applications as e.g. surface plasmon resonance (SPR) or biolayer interferometry (BLI)

Furthermore, higher protein yields compared to Strep-Tactin® are obtained. On average, StrepTactin®XT provides almost 2-fold more protein than Strep-Tactin®.

Strep-Tactin®XT also ensures sharp elution profiles achieving high concentration of the target protein. Strep-Tactin®XT even tolerates a wide pH range in addition to various detergents and additives, contributing to the universal usability of the system.

Resin reuse

Strep-Tactin® and Strep-Tactin®XT resins for protein purification can be regenerated and reused 3 to 5 times without loss in performance. The proper regeneration of the column and resin activation can easily be checked with HABA. The yellow HABA solution turns red (Strep-Tactin®) or orange (Strep-Tactin®XT) upon binding  to the engineered biotin binding pockets of Strep-Tactin® and Strep-Tactin®XT clearly indicating that the resin is fully regenerated. Afterwards, HABA can be removed by washing with 1x Buffer W. Once the red color has disappeared the column can be reused.  If the biotin binding pocket is blocked or damaged no color shift occurs and the resin cannot be reused.

Thumbnail for Sepharose resin regeneration check video

What is the difference between Sepharose®, Superflow® and 4Flow®?

The underlying matrix of many protein purification resins are porous agarose beads. The characteristics of these agarose beads determine for which proteins and for which applications the resin is most suitable. IBA’s Sepharose® resin consists of a 4% cross-linked agarose and is therefore suitable not only for small, but also for large proteins. However, a disadvantage of Sepharose® is that it is not pressure stable and consequently not suitable for automated applications such as FPLC. In contrast, IBA’s Superflow® products work well for FPLC. The disadvantage of Superflow® is that it consists of a 6% cross-linked agarose. This means that the pores are smaller and not accessible for larger proteins, resulting into low yields.
4Flow® combines the advantageous characteristics of Sepharose® and Superflow®. It is a 4% cross-linked agarose, and it is pressure stable, making it a universal matrix for small and large proteins as well as for different applications such as FPLC.

Specifications for Strep-Tactin® Purification Resins 

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.
 

Strep-Tactin® 

Strep-Tactin®XT

Matrix Superflow®  Sepharose® MacroPrep® 4Flow® MagStrep Strep-Tactin®XT beads
      (only available as 20 ml slurry)    
Binding capacity*/
Dynamic Binding capacity**
classic: 3 mg/ml resin* classic: 3 mg/ml resin* classic: 3 mg/ml resin* classic: 5 mg/ml resin** classic: 25.5 µg/µl resin*
  high capacity: 7.0 mg/ml resin**     high capacity: 14 mg/ml resin**  
Bead structure 6% agarose, crosslinked 4% agarose, crosslinked polymethacrylate 4% agarose, highly crosslinked magnetic core covered with 6% agarose, crosslinked
Bead size 60-160 µm, spherical 45-165 µm 50 µm 50-150 µm, spherical 30 μm (average), spherical
Exclusion limit 6 x 10Da ~3 x 107 Da 1 x 106 Da 3 x 107 Da not specified 
Recommended  flow rate 0.5-1 ml/min  gravity flow 0.5-1 ml/min  0.5-1 ml/min batch purification
pH range for protein binding 7-8 7-8 7-8 4-10 6-10
Max pressure 9.6 bar gravity flow 70 bar 3.5 bar batch purification
Storage 4 °C, do not freeze 4 °C, do not freeze 4 °C, do not freeze 4 °C, do not freeze 4 °C, do not freeze
Shipment RT RT RT RT RT
Eluent desthiobiotin
desthiobiotin
desthiobiotin
biotin biotin
Regeneration buffer Buffer R Buffer R Buffer R Buffer XT-R 100 mM NaOH
Features and recommendations
  • Strep-Tactin® Superflow high capacity binds three to five times more protein in comparison to Strep-Tactin® Superflow
  • Can be used for the purification of biotinylated proteins
  • For average sized proteins (<90 kDa)
  • For proteins that are hard to elute from Strep-Tactin®XT resins, like proteins with multiple tags or multimers
  • Suitable for use in FPLC columns
  • For large proteins (>90 kDa)
  • Can be used for the purification of biotinylated proteins
  • For proteins that are hard to elute from Strep-Tactin®XT resins, like proteins with multiple tags ore multimers
  • Only suitable for gravity flow due to low pressure stability
  • Synthetic support
  • Reduced unspecific binding
  • Strep-Tactin®XT 4Flow® high capacity binds three to fove times more target protein compared to Strep-Tactin®XT 4Flow®
  • Applicable for all protein types, especially large (>90 kDa), low abundant and challenging proteins
  • Strep-Tactin®XT 4Flow is recommended for first time users of the Strep-tag® system
  • Optimized for Batch purification
  • Beads can be separated with a magnetic device, no centrifugation necessary
  • Suitable for purification in 96-well plates

* Max binding capacity for a Strep-tag® protein (30kDa).

** Dynamic binding capacity (DBC) was determined with mCherry-Twin-Strep-tag (30 kDa) under realistic operation conditions and shows the amount of protein which is bound until 10% of the protein is found in the flow through.

Find out more about Strep-tag® protein purification

Here, we have grouped links to make it easier to find more information about Strep-tag® technology for protein purification. In our web shop, you can find the protein purification products  that fit your research needs. Read the comprehensive comparison of the most used tag systems, Strep-tag® vs. His-tag. Browser through application examples and frequently asked questions about Strep-tag® purification system or download application notes in our download area.