1. Raise a polyclonal antibody to the purified protein and do a Western blot on cells expressing the protein.

2. Alter the gene by incorporating a peptide tag at one end for which you can buy an antibody, such as FLAG, then use that antibody for the Western blot.

HI Adam,

Thanks for your suggestions. However, we already have a HA tagged protein being expressed and while using an anti HA antibody in western blot we see three bands. I just need another supplementary assay to confirm that the bands in western are not artifacts but result of actual processing event.

Dear Sumit

probably the simplest things is replace the tag e.g with an his and repeat the esperiment. if you see the same fragments with an antibody with different specificity, than your fragments are probably real fragments.

one other possibility is try to generate an inactive mutant of the protease ( if you produce an ha tagged form i suppose that you know the sequence) by replacing a catalitic residues and try to inactivate it to undertand id degradation is auto-proteolysis.

of course one other possibility if you are able to purify a small amount of the fragments (eg replacing ha with his tag) is transfer the purified band in pvdf and send it to n-term sequencing by edman degradation that in case it is a fragment of your protease will provide to you Also the sequence of the fragmentation site.

https://www.alphalyse.com/sequencing/n-terminal-edman-sequencing/

just a question; are you producong it in e.coli? in mammalian? in its original host?

good luck

ciao

Manuele

I'm not really sure what you mean by "gets processed". I speculate that there may be autoproteolysis of the protease, in which case digestion might increase with time and the full length protein might all but disappear. Also, the sizes of the fragments might give you a rough idea of the targeted peptide sequences (Manuele's suggestion above is better!) . It might also be that another protein, serum albumin for example, would be degraded by the protease, although I'm not sure if that would address your question!

My experience with tagged proteins (not HA, admittedly) suggests that the tag can get cleaved off by host proteases, but I dont recall examples of truncated proteins that retained the tag.

If you are expressing the protein by using some vector, you can try with different cells and check the protein expression by western blot analysis. It can give you some kind of idea whether they are actually an artifact or processed proteins.

3. Perform an immunoaffinity purification of the protein using the anti-HA antibody, then run the purified protein on SDS-PAGE. If you get bands that are in sufficient quantity to see with Coomassie Blue staining, cut them out and submit them for mass spectrometry sequencing for identification.

Did you try western blot of the whole cells? Simply centrifuge the cells, ressuspend in loading buffer, boil and load on gel.

A good control could be eampty vector.

@ Manuele: That's exactly what we are planning to do...however, mass spec. is costly. So I was wondering if there is any quick and dirty expt. that could be done...But thanks again for the useful suggestion. Also, we have already generated the catalytic mutant and it still gives three bands. What we believe is probably there is another protein which is responsible for it's cleavage. In other words our protein of interest does autoprocess itself.

@ Sailesh: Yes, we expressed in other cell lines (both mammalian and insect cell lines) and we do see the same three bands as in our original cell line

Dear Sumit,

Have you tried native gel for Western blot. Sometime, denaturing conditions also can affect protein cleavage. Therefore, you can try native PAGE and see whether you still get leaved protein bands.

Thank you.