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

The Strep-tag® system is one of the most widely used affinity chromatography systems for protein purification, detection and immobilization, and offers many advantages.

Besides the classical Strep-Tactin® resin, IBA also offers an improved version called Strep-Tactin®XT.

10 reasons why Strep-tag® is the leading technology in protein purification
  1. Unparalleled protein purity (> 95 %).
  2. Physiological purification conditions preserve functionality.
  3. Time saving due to a fast one-step purification requiring low washing volume.
  4. Variability in buffer conditions, e.g. high salts, detergents, metal ions, chelators or reducing agents.
  5. High specificity of Strep-tag®:Strep-Tactin® with competitive elution using desthiobiotin enables low background.
  6. Re-usability of the robust purification resins.
  7. Favorable for protein:protein interaction studies due to mild elution conditions and low washing volumes.
  8. Assay implementation via immobilization of Strep-Tactin®XT (reversible binding) or StrepMAB-Immo (irreversible binding).
  9. Removal of the small tag is not necessary since it has a neutral pI and does not influence protein folding or function.
  10. Availability of a universal detection system for Western blot, ELISA, Immunofluorescence, FACS and more.
Key features of the Strep-tag® system
  • Strep-tag®II is a very short peptide tag with only 8 amino acids (Strep-tag®II sequence:  SA-WSHPQFEK)
  • Rapid one-step purification under physiological conditions
  • Unsurpassed purity and bioactivity of target proteins
  • Balanced/inert amino acid composition - no known effect on protein structures or activities
  • Highly selective binding properties
  • C- or N-terminal fusion
  • Removal of tag is not required
When should the Strep-tag® 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
  • and any other protein
Strep-tag® and Twin-Strep-tag®

Strep-Tactin® can be used in combination with the following tags:


  • 8 amino acids
  • sequence: Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (WSHPQFEK)


  • Two Strep-tag®II motifs in series
  • Higher affinity than Strep-tag®II

Features of both tags

  • Fusion to the N- or C-terminus
  • No influence on protein folding and function
  • No removal necessary
Strep-tag® principle

Strep-tag® principle

The basis for the development of the Strep-tag® system is the well known binding of biotin to streptavidin. To take advantage of this very strong interaction in protein purification applications it was necessary to investigate a peptide that is capable of binding to the biotin binding pocket of streptavidin when fused to recombinant proteins. This peptide was supposed to serve as purification tag. The found peptid was a short sequence consisting of only 8 amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) and was named Strep-tag®II.

In order to optimize the binding properties, also streptavidin has been engineered to obtain Strep-Tactin®. Thus, the optimal binding partners have been found: The Strep-tag®:Strep-Tactin® system is now one of the most widely used affinity chromatography systems.

The Strep-tag® purification system is based on the highly selective and easily controllable interaction between the Strep-tag®II peptide and Strep-Tactin®. The binding affinity of Strep-tag®II to Strep-Tactin® is nearly 100 times higher than Strep-tag®II to streptavidin. The tagged protein binds to the immobilized Strep-Tactin® during the affinity purification process. Physiological buffers like PBS in combination with a wide range of additives can be used (see Table 1, Strep-Tactin® resin specifications). After a short wash step, gentle elution of purified recombinant protein is performed by addition of desthiobiotin (2.5 mM) in 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 thus enabling unparalleled purification factors. The system is safe and easy to use; column regeneration and activity status are visualized by a color change on the purification column.



The short peptide tag (8 amino acids) has only negligible effects on the recombinant protein due to its chemically balanced amino acid composition (WSHPQFEK). The tag can be placed at the C- or N-terminus. A two amino acid spacer between the protein and the tag is recommended to ensure accessibility of the tag.

Benefits of Strep-tag®II:

  • it does not interfere with folding or bioactivity
  • does not react with heavy metal ion buffer impurities
  • has no ion exchange properties
  • does not induce protein aggregation
  • no need for removing the tag


Strep-Tactin® is a derivative of streptavidin, which is one of the most stable proteins known.


  • Purification of bioactive recombinant proteins
  • Physiological purification using desthiobiotin elution
  • Protein aggregation is avoided
  • Broad range of detergents, chelators, salt or redox conditions allowed
  • Avoids interaction with heavy metal ions which are toxic and may catalyze protein oxidation
Strep-tag® purification cycle

Purification procedure under physiological conditions

The purification of Strep-tag®II fusion proteins is easy, straightforward and user-friendly. The complete procedure can be performed under nearly physiological conditions, e.g. in PBS buffer and for elution in PBS/2.5 mM desthiobiotin buffer:

Steps 1 + 2: The cell lysate is added to the column. Once the tagged protein has bound specifically to Strep-Tactin® the host proteins are washed away rapidly with small amounts of physiological wash buffer (Buffer W).

Step 3: Bound Strep-tag®II fusion proteins are then gently eluted by adding wash buffer supplemented with 2.5 mM desthiobiotin (Buffer E), which specifically competes for the biotin binding pocket.

Since the buffer conditions during elution essentially remain unchanged, potentially unspecifically bound proteins (without Strep-tag®) will not be eluted and, thus, will not contaminate the protein of interest. Next to the specific binding of Strep-tag® to Strep-Tactin®, this is the second specificity conferring step of this purification procedure, yielding extremely high protein purity.

Steps 4: To regenerate the column the yellow azo dye HABA (2- [4’-hydroxy-benzeneazo] benzoic acid) is added (buffer R) in excess to displace desthiobiotin from the binding pocket. Once HABA binds to the binding site, the color turns to red conveniently indicating the regeneration and activity status of the column.

Step 5: HABA can be removed simply by adding wash buffer. Once the red color has disappeared the column can be re-used. Strep-Tactin® resin can be regenerated and re-used 3 to 5 times without loss of performance.

Purification of a GFP-Strep-tag®II fusion protein, which has been overexpressed in E. coli.
Pictures left to right:

1 - New or regenerated column

2 - Specific binding of GFP-Strep-tag®II fusion protein to Strep-Tactin® Sepharose® column while unspecific proteins are rapidly washed away with small amounts of physiological buffer

3 - Strep-tag®II protein is eluted due to addition of the specific competitor "desthiobiotin"

4 to 6 - Column regeneration: desthiobiotin is displaced by the yellow solution HABA, which turns red once complexed with Strep-Tactin®. HABA is then removed by washing buffer and the column can be re-used

Learn more about the Strep-tag® technology in our free tutorials.

Strep-tag® tutorials