How does a Strep-tag work?
How does a Strep-tag work?
As the Strep-tag binds reversibly to the same pocket where the natural ligand D-biotin is complexed, it can be applied for the efficient purification of corresponding fusion proteins on affinity columns with immobilized streptavidin5.
What is strep Tactin resin?
Strep-Tactin ® Sepharose ® is a 4% agarose coupled with the streptavidin variant Strep-Tactin ® and applicable for purification of Strep-tag ®II or Twin-Strep-tag ® fusion proteins.
What is Desthiobiotin?
Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by biotin. We synthesized an amine-reactive desthiobiotin derivative for labeling proteins and a desthiobiotin-agarose affinity matrix.
How do you regenerate Tactin resin for strep?
After purification, the resin can be regenerated with Buffer XT-R or alternatively with fresh NaOH solution (10 mM) and reused three to five times without loss of yield.
How do you purify strep-tagged proteins?
Agarose-based matrices with the coupled Strep-Tactin ligand are the resins of choice, with a binding capacity of up to 9 mg ml(-1). For purification of lower amounts of Strep-tagged proteins, the use of Strep-Tactin magnetic beads is suitable.
What is Ni NTA resin?
Ni-NTA Agarose is a nickel-charged affinity resin that can be used to purify recombinant proteins containing a polyhistidine (6xHis) sequence. Proteins bound to the resin may be eluted with either low pH buffer or by competition with imidazole or histidine.
How do you dissolve Desthiobiotin?
D-Desthiobiotin is sparingly soluble in aqueous buffers. For maximum solubility in aqueous buffers, D-desthiobiotin should first be dissolved in DMSO and then diluted with the aqueous buffer of choice. D-Desthiobiotin has a solubility of approximately 0.2 mg/ml in a 1:2 solution of DMSO:PBS (pH 7.2) using this method.
Does Strep Tag bind biotin?
The strep-tag has the property of binding to streptavidin competitively with biotin. This behavior permits the use of very gentle conditions for the elution of a bound strep-tag fusion protein from the streptavidin affinity column, just by applying a diluted solution of biotin or one of its chemical derivatives.
What is Twin strep tag?
Twin-Strep-tag® (TST) binds to a single Strep-Tactin® tetramer. • TST arrangement provides enhanced affinity by off-rate reduction. • TST binds reversibly and enables efficient competitive elution.
What is Tactin?
Strep•Tactin® protein, a derivative of streptavidin, has an optimized binding pocket that is specific for the Strep•Tag II peptide. It is a stable protein that has chemical properties similar to those of streptavidin, making it compatible with a broad range of detergents, chelators, salt, and redox conditions.
How do you clean Ni-NTA resin?
For Qiagen’s Ni-NTA, a simple regeneration protocol is:
- Wash with water.
- Remove Ni2+ ions with 50 mM EDTA.
- Wash with water.
- Clean with 0.5 M NaOH.
- Neutralise with water (this will take some time)
- Regenerate with 100 mM NiSO.
- Wash with water and then either 20% ethanol or buffer.
