Hi to all,
Is there any method mass-spec compatible to extract proteins from an agarose gel and purify them so that I can identify them by a direct injection in a ESI-MS?
For example if I dissolved Agarose by heating and I performed a zip-tip purification on the dissolved selected band, Would I achieve my goal?
Thanks
You might want to check this one.
http://www.ncbi.nlm.nih.gov/pubmed/18048052
Try separating your protein on a polyacrylamide gel. I have never heard of someone using an agarose gel. There are standard protocols for extracting protein from polyacrlylamide gels. Just google for "protein extraction polyacrylamide". For example http://www.ncbi.nlm.nih.gov/pubmed/15765489
I know how to extract protein from pas gels, i' d like to know if someone knows how to extract them from agarose gel
You might want to check this one.
http://www.ncbi.nlm.nih.gov/pubmed/18048052
thank you very much, I think this article will be very useful for my purposes
Something I heard about many years ago but never tried: Excise the the protein band from the agarose gel. Place inside a Costar 'Spin X' column (or similar), chop with a spatula (be careful not to damage the membrane), put at -20C for 10 min, and centrifuge at micro-centrifuge 13,000 x g for 5 minutes at room temperature. Your protein should be at the bottom tube. Now precipitate or use ultra-filtration to exchange buffer as needed.
As a starter, to save money, you can try using a 200ul eppendorf tube inside a 1.5 ml tube, pierce a few holes in the 200 ul tube bottom using a needle.
Good luck and if you do try please let us know if it works!
Yoram
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