Hi everydody,
I try to purify à protein with a N-terminus strep tag follow by à thrombin cleavable site using strep tactin resin (IBA). This protein can be purify using a His-tag in the same place... So the tag is accessible
I already use this resin to purify a soluble protein... so the resin is functional. Regarding my membrane protein, I use DDM 2% w/v in HEPES 50 mM pH 7,5, NaCl 500 mM, glycerol 10% v/v for the extraction of my protein. MY purification buffer is HEPES 50 mM pH 7,5, NaCl 300 mM, glycerol 10% v/v DDM 0,02% w/v. I perform the elution with 2,5 mM of desthiobiotin.My protein is detected in the membrane extract.. But no binding is observed.
Does anybody have a idea to fixe my problem?
Adrien.
Perhaps the DDM concentration in the purification buffer (0.02%) is too low to keep the protein in solution.
I use 0.02% of ddm when I perform IMAC with the his-tagged form of this protein... so I don't think that is the problem..
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.
I have 2 distinct plasmids... one to express a his tagged form of my protein A and another where the his tag is replaced by a strep tag II. The IMAC works with first plasmid. But I need to purify the strep tagged form also... and it doesn't work as previously mentioned.
Hello Kariona,
I see my protein (in fact it's a membrane tri-complex) in the flow through so it's a problem of binding... I'll try to change de pH and may be dilute my extract to reduced the quantity of detergent...
thanks for your answer.
Dear Adrien,
Try StrepTactin XT from IBA. It has a higher binding affinity to Strep-tagII compared to Strep-Tactin and tolerates higher detergent concentrations. They also provide a free Trial Kit, if you ask for this. Contact: [email protected].
Hi everybody,
I finally purify my protein complex at pH 8 and by dilution of my sample after membrane extraction (0,5% DDM final)... Nervertheless a large amount of my protein is in the flow through and it is not due to resin saturation... I'll maybe try twin-streptag that is more suitable for detergent...
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