I am over expressing my protein which has a 6x his tag, using lentiviral infection in a mammalian cell line. I also have a reporter in my lentiviral construct which gives me an idea of transduction efficiency.
The his tag is at the N-terminus and there is a thrombin cleavage site after it.
I am able to detect the overexpression using gene specific antibody and not the anti-6X his antibody.
My first conclusion was the antibody was not working. But is it possible that the his tag might have fallen off/cleaved? Or 6 histidines were not sufficient to be detected by the antibody? Or his tag epitope was not accessible for the antibody?
I denature my samples at 70 degree celsius for 10 minutes and this is the standard protocol followed in the lab (also as per life technologies loading dye). Does anybody think this temperature may also cause the tag to fall off?
I am also going to overexposes my protein with 6xhis tag at the C-terminus and see if that works.
Appreciate for your time and effort.
It is unlikely that the heating caused cleavage of the His tag. And 6 His is sufficient for any good His antibody to work. If you can get a different His tagged protein as a positive control it would likely help in your analyses. It is certainly possible that the N--terminal His tag was cleaved in the cell. But be aware that some small tags, including His6 can be masked by the context and not show up in immunoblots. Even though you denature proteins by heating and SDS some proteins can at least partially fold and prevent access to the tag. I have seen this in one instance with my proteins, though it is quite rare. You could also try putting the His6 tag on the other end, if that doesn't interfere with activities.
It is unlikely that the heating caused cleavage of the His tag. And 6 His is sufficient for any good His antibody to work. If you can get a different His tagged protein as a positive control it would likely help in your analyses. It is certainly possible that the N--terminal His tag was cleaved in the cell. But be aware that some small tags, including His6 can be masked by the context and not show up in immunoblots. Even though you denature proteins by heating and SDS some proteins can at least partially fold and prevent access to the tag. I have seen this in one instance with my proteins, though it is quite rare. You could also try putting the His6 tag on the other end, if that doesn't interfere with activities.
Some of the early His tag antibodies were actually directed to other amino acids in the tag sequence and not the His residues, which are not typically very antigenic. More modern His tag antibodies should detect the 4-8 poly His region if run as the Mfg. recommends. Do you have any positive controls? I've found that recombinant TEV protease commonly has a His tag and can be a good control lane. Proteaolysis that specifically cuts the His tag off is rare unless a cleavage site was included, general proteolysis will usually cleave multiple places. Mammalian cells can have thrombin like activity, perhaps check this and maybe include a specific inhibitor.
Proteins that "tuck their tail/head" and occlude a C or N terminal tag are known but rarely maintain this behavior in SDS-PAGE Western blotting, I'd suspect the antibody and include a positive control (Occam's razor!) Good luck!
I agree that you should test running a His-tagged positive control as well, to verify that the antibody detection is indeed working. You say that you were able to detect the protein using gene-specific anybody instead, how did that blot look? Did the protein come out at the expected size, or does it appear smaller than it should? In that case you could suspect that there has indeed been a proteolytic cleavage of the His-tag.
Thanks to everyone for all of the suggestions. My next approach is to check with a positive control, check with c-terminal his tag. If the problem persists than will have to use a thrombin inhibitor added to protease/phosphatase inhibitor cocktail.
Are you sure the His tag and your complete protein are both there? Was genetic sequence checked? Peptide fingerprint analysis done?
Also, as others have said, always do a positive control. You can also try a His stain (we use InVisionTM in our lab--it's relatively cheap and very easy . . . but not as sensitive as western blotting, of course). Also- don't skimp on the protease inhibitors just in case it is being degraded, etc. Good luck!
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