I have been trying to purify a GST-tagged eukaryotic protein from E. coli pLysS. I am getting it in low concentration along with a big blotch of non specific proteins.
Are you able to be a bit more specific or show a gel? Was the protein highly expressed in the induction?
Which vector are you using? Can you cleave your protein off the GST tag instead of elution?
What is the "big blotch" of non-specific proteins? E.coli chaperone proteins can sometimes be significant contaminants of GST-tagged proteins, especially if the fusion protein is mis-folded.
Some more information about your experiment would be helpful if you would like directed advice.
Best wishes.
Your induction temperature, time, the vector you are using, washing steps during purification---all of these factors should be looked upon individually
I assume you are referring to the SDS-PAGE analysis of the flow through from the affinity column when you write " NON-SPECIFIC protein" and therefore the big blotch is the flow through when loading the column? So the "low concentration" is referring to the fusion protein seen on the gel analysis of the column eluate? At this point is the "low concentration" material relatively impure and and you want to know how to purify it further or do you want to know how to increase expression level?
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