This protein is GST-Tagged and also soluble. But I'm facing problem in the purification step.PBS buffer pH7.3 as a binding buffer and Tris-HCl + L-Glutathione (reduced )pH.8. this protein eluted with elution buffer along with other proteins. kindly suggest me how to improve purification. I have attached SDS-PAGE result.Thanks in advance.
Hi Ali,
This may be due to poor affinity of fusion protein with the glutathione Sepharose. You can try using mild lysis approach may be your protein denatures with the conditions you are lysing your cells. There's an good article you can refer. Please follow the link
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584333/#FN26
Thanks.
Hi Farman,
After you passed the supernatant through the column, did you wash the column with binding buffer? If yes, you could include a high salt wash (binding buffer with 500 mM NaCl).
If that large band is your elution fraction, it doesn't look too bad. There are some lower molecular weight bands, quite possibly from degradation, which you may be able to reduce by adding in protease inhibitors if you haven't already.
Other than that, wash your column with a larger volume of binding buffer, e.g. 50 CV, and as Nicholas states you could try a high salt wash. Other things to try would be to proceed to further purification steps. A combination of ion exchange and gel filtration will probably get you a very pure sample.
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