How can we help you? ...Answers to questions about the Strep•Tag®/Strep•Tactin® Affinity Purification Products What is the principle of Strep•Tag® technology?
The Strep•Tag purification system is based on the highly selective and easily controlled interaction between the Strep•Tag® II peptide and Strep•Tactin ®, a specifically engineered streptavidin. The tagged protein binds to immobilized Strep•Tactin during affinity purification. Physiological buffers, like PBS, in combination with a wide range of additives can be used. After briefly washing, purified recombinant protein is gently eluted by adding 2.5 mM desthiobiotin 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, which yields unparalleled, one-step purification. Column regeneration and activity status are visualized by a color change on the purification column. The system is safe and easy. What is the size of Strep•Tag II and where can it be placed on the fusion protein?
Strep•Tag II has eight amino acids (WSHPQFEK) and a molecular weight of 1 kDa. It can be attached to the N- or the C-terminus or between two protein domains as a linker, or even used in loop structures. What degree of purity can be expected?
Over 95 %. However, impurities resulting from non-specific interactions with the recombinant protein itself could lead to lower purity. To reduce contaminants covalently linked to the recombinant protein by disulfide bonds, add reducing agents to all buffers for cell lysis and chromatography. For non-covalently linked contaminants, increase the ionic strength in all buffers for cell lysis and chromatography by adding NaCl or mild detergents (i.e. Triton X-100, Tween 20, CHAPS). Does Strep•Tag II bind avidin?
No. Therefore, avidin can be used to block naturally occurring biotinylated proteins. For proteins expressed in the cytoplasm, is the presence of biotinylated proteins in the host organism a problem?
No. Generally, the amount of biotinylated proteins in the cytoplasm is very low and does not lead to significant inactivation of the column. In an E. coli extract derived from a 1 L culture with OD 550 = 1, the total biotin content is only around 1 nmol; column capacity is 350 nmol/ml. Even the biotinylated E. coli protein BCCP has a relatively low intracellular concentration and usually does not interfere with purification. To avoid binding BCCP to Strep•Tactin resin, add avidin to the cell lysate before chromatography (20 mg/L for an E. coli culture at OD 600=1). For proteins secreted into the medium, is the presence of free biotin in the medium a problem (eukaryotic expression)?
The amount of biotin depends on the cell line and the medium. Some media for insect cells or mammalian cells contain up to 800 nmol biotin per litre and has to be removed or blocked before applying the lysate to Strep•Tactin columns. For mammalian cell culture only DMEM and Leibovitz's L-15 media are free of biotin. For insect cell culture, only Schneider's medium is free of biotin. Serum may also contain biotin. However, serum tested (FCS, PAA) did not contain measurable amounts of biotin (<0.025 mg/ml; <0.1 mM). Ingredients of proprietary formulations for serum free growth are usually not disclosed, but information on biotin content can be obtained and should be requested from the respective manufacturer. When the protein elutes from the column, is it complexed with desthiobiotin?
No. Desthiobiotin does not complex or interfere with the protein or general protein assays and can be removed by gel filtration or dialysis. Is the bioactivity of the protein preserved?
Yes. The physiological buffers used for washing and elution preserve the bioactivity of the protein. Is it possible to detect protein-protein-interactions using Strep•Tag technology?
Yes. The One-STrEP TM Protein Interaction Kit (Cat. No. 71624-3) was specifically designed to isolate intact protein complexes. Is there a convenient method for parallel purification of different Strep•Tag proteins?
Yes. The Strep•Tactin ® HT96 Purification Kit (Cat. No. 71605-3) was designed for automated, high-throughput purification of Strep•Tag proteins (up to 100 m g Strep•Tag protein per well). The plates are pre-loaded with Strep•Tactin affinity resin and simply have to be rehydrated and equilibrated before use. The plate can be used with standard vacuum manifolds for manual sample processing or is compatible with robotic sample processing systems, such as the Genesis RSP/RWS Separation System (Tecan), the MultiPROBE II (Packard BioScience), the BioRobot workstations (Qiagen) and the Biomek 2000/FX robots (Beckman Coultier). Can the Strep•Tag be cleaved off?
Yes. There are vectors containing enterokinase or HRV 3C cleavage sites. However, due to the small size and chemically inert nature of Strep•Tag II, it generally does not interfere with the folding or bioactivity of the recombinant protein and does not need to be removed. Can detergents or other buffer systems be used?
Yes. As long as the pH remains above pH 7.0, high salt, reducing reagents like mercaptoethanol, chelating reagents, and detergents can be used with Strep•Tactin, but usually are not necessary. The following reagents have been successfully tested. How, and how many times, can Strep•Tactin be regenerated?
The matrix is regenerated with an azo dye, hydroxyl-azophenyl-benzoic acid (HABA), which, when applied in excess, displaces desthiobiotin. The dye is yellow in solution and shifts to red when bound by Strep•Tactin, allowing visual control of the regeneration process and the functional status of the column. As long as a color gradient between the top and the bottom of the column is visible, it is not fully regenerated. The resin can be regenerated 3 to 5 times. How can Strep•Tag fusion proteins be detected?
For high specificity detection, Strep •Tag II monoclonal antibodies, either conjugated to HRP (Cat. No. 71591-3) or without a conjugated enzyme (Cat. No. 71590-3), are available.
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