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[ana4]

I have cloned and expressed my protein in BL21DE3 cells using pGEX-4T2 vector and have succesfully purified the recombinant protein using affinity column(glutathione). but when i tried cleaving the GST tag with the RECOMT thrombin cleavage kit(sigma), i didnot see any cleavage of the tag from the protein as seen on the gel. Have been facing this problem and have not yet been able to obtain protein without the tag. I have carried out the reaction at around 25 degrees as mentioned in the kit for 24 hrs but with no result. Please can anyone tell me if u have used some other technique or kit for performing the GST tag cleavage?

[bob1]

How big is your protein?  It may be that it has cleaved, but you didn't see a size shift because the protein is too big relative to the GST.