Sequencing protein after SDS-PAGE

[radioheadhead]

Hello all,


I need to have a protein sequenced, and I plan to separate it from other proteins by SDS-PAGE. I understand that it is possible to then have the protein sequenced off of the PVDF membrane, how would one go about this! Any suggestions or links are appreciated.

Thanks,
toby

[danfive]

Either stain gel with MS-compatible stain then send in the gel slice containing the band. If you want to transfer to a membrane (like for Western Blot) then proceed as normal. Either way keep everything sterile and keratin free, as a contaminating protein can mask/obscure your isolated protein.
Remember 2D separation is best when multiple proteins are likely to be present at a given band in 1D SDS-PAGE.

[danfive]

Also recommend trypsinization and LC-MS-MS, it can give you more peptide separation; stay away from PMF, peptide mass fingerprinting, usually on a MALDI-MS (just isn't good enough).

[danfive]

If you're contracting out for MS services I recommend these guys --> http://www.prsproteomics.com/service...ectrometry.htm

I've used them, they are very knowledgeable and very helpful.

[radioheadhead]

Thanks all! I am not set up for 2-D, but I do have an antibody to my protein of interest so I will try to get it as pure as possible with a protein A column to minimize other proteins at the same (95kD) molecular weight.


Quick question, if I bind IgG to a protein A column, then run my lysate through to retain my p95 target, is it possible to elute just my target protein without the antibody? Or will I need to elute both and then separate them? I am, of course, going to boil in SDS before PAGE, so I guess this would separate the mAb-p95 complexes, correct?

[danfive]

I did the exact same thing and it worked well. I bought a kit that fixes/crosslinks the capture antibody to protein G beads. Then you perform the capture step, wash extensively and elute only your protein of interest. The kit is called Seize X Protein G Immunoprecipitation Kit from Pierce, it comes with either Protein A or G beads.
Just be aware that the Protein A or G will bind any native antibodies in your lysate (or in my case biological fluid) and those antibodies will likely elute and contaminate you immunopurification.
Also albumin and other major proteins may not wash out with a normal amount of washes (like recommended in std protocol) but I was able to get rid of albumin by increasing wash step to 25-30 times instead of 4-8 times.

Of course if you don't crosslink your antibody it will co-elute.

[radioheadhead]

Originally Posted by danfive

I did the exact same thing and it worked well. I bought a kit that fixes/crosslinks the capture antibody to protein G beads. Then you perform the capture step, wash extensively and elute only your protein of interest. The kit is called Seize X Protein G Immunoprecipitation Kit from Pierce, it comes with either Protein A or G beads.
Just be aware that the Protein A or G will bind any native antibodies in your lysate (or in my case biological fluid) and those antibodies will likely elute and contaminate you immunopurification.
Also albumin and other major proteins may not wash out with a normal amount of washes (like recommended in std protocol) but I was able to get rid of albumin by increasing wash step to 25-30 times instead of 4-8 times.

Of course if you don't crosslink your antibody it will co-elute.

ahh, thanks, that was what I needed. I already have some protein A columns that I plan to apply my IgG to, I will just need to cross-link it to the protein A via chemical means before running my protein sample through. My lysate is from breast cancer cell in vitro, so extraneous antibody will not be a problem.

[danfive]

The kit I used employed DSS crosslinker, here's the manufacturer info --> http://www.piercenet.com/Products/Br...fldID=02030236
Hope it is useful.

[radioheadhead]

great! I think I'm gonna try the column-based one with DSS, that way I will have a reusable capture column for my protein. I'll let you know how it goes!