Science Buddies: "Ask an Expert"

I have posted my questions here a few times before and have gotten a lot of great help. I am still working on my project of sequencing a myoglobin protein. Well I managed to get my SDS page bands digested at a facility near me and mass spec'd + fingerprinted, and the results, while good, only resulted in 81% coverage of my entire sequence. Since they only do tryptic digests I'm faced with the task of doing an alternate digest myself to increase coverage. Based on my theoretical sequence derived from the DNA, I've chosen Glu-C endoproteinase as my enzyme (with Asp-N as a backup).

Does anyone have a simple and good protocol for a Glu-C in-gel digest? I am worried I won't have access to the kind of equipment I may need such as speed-vacs, agitated water baths, and the like.

I've thought about getting a kit like such: http://www.piercenet.com/product/in-gel ... estion-kit

But I don't know if that's necessarily worth it. I'd still need to get some Glu-C because it's a tryptic kit.

Do I really need to alkylate and/or reduce? I've read this is for cysteine residues and my sequence shouldn't have any cysteines I believe.

Sorry for all the questions. I wasn't planning on having to do my own in-gel digest so now I'm scrambling to get it all together.

Thank you

I have used V8 protease [Glu-C] to cleave proteins in solution but never in a gel. It worked acceptably, but don’t over-digest because the enzyme can also cleave at aspartates.

You are correct about not needing alkylation if there are no Cys in your peptide.

Have you tried calling New England Biologicals? They have really good tech support and may be able to give you some tips on in-gel cleavage reactions.

Good luck,

Sybee