Hi everyone,
I have a problem regarding the purification of a protein using GST system. My protein is expressed well attached to GST in E.coli Bl21. After lysis and loading on glutathione sepharose resins and several washes the GST-protein band is detectable on SDS-PAGE. However, on-column cleavage of protein from GST using prescission results in no protein in the elution. After that using glutathione the GST is recovered from column with no protein attached to it. I was wondering what was happened to my interest protein during prescission activity. I have done the prescission cleavage based on the standard protocol at 5 C for 16 h.
Ok, this sounds indeed strange. But please clarify a few things for me:
First, by "GST-protein band" band you mean that you find the fusion-protein on the beads and can detect them via SDS-PAGE. 26kDa + The size of your protein
And after protease cleavage you can only detect GST anymore at 26 kDA?
Dear Sebastian Lühn,
Thanks for your help. Yes, exactly happens what you said.
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