Hello all,
I am facing an annoying problem wherein I am attempting to remove my GST tag from my (glutathione eluted) chimeric protein using a Thrombin Cleavage kit (Biotinylated Thrombin), but however after 10 hours of cleavage reaction at 20 degrees I am not being able to get the protein in the supernatant , but however it is remaining bound to the glutathinone sepharose beads.
I need it in the supernatant for use. The protein w/o GST tag is of ~17kda.
WHY IS MY PROTEIN OF INTEREST, without the GST TAG interacting with the glutathinone sepharose beads?
NB: The vector I am using is pET42 which has a thrombin cut site upsite of my protein.
I have attached an image of sds where there is LtoR : marker , supernatant post incubation of eluted protein with thrombin , and glutathinone sepharose beads incubated with thrombin treated chimeric protein. sorry for bad quality image
Hi @Rajarshi Roy... as your cleaved protein non-specifically binding with the Glutathione matrices you may try size-exclusion chromatography if native molecular sizes of your cleaved protein and GST tag are having differences.
Ravi Sanyal hello Sir, thank you for your response. I was thinking size exclusion myself , but however I was also wondering why is my cleaved protein binding with the gst beads? I am washing them after cleavage but however I am not getting it in the supernatant. what could be going wrong?
@Rajrshi Roy if after cleavage protein is not present in supernatant that shows protein is having some folding issue and GST tag makes it remain soluble. That also clarifies why your protein comes with GST beads. To further confirm this you may try in solution digestion and after a high centrifugation load the supernatant and see which fraction your cleaved protein present.
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