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SDS-PAGE, please help!!!


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#1 anonymous

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Posted 04 September 2001 - 09:00 PM

I have purified two isoforms of trypsin from anchovy. Confirmed by SDS-PAGE, N-terminal aa sequencing and substrate specificity and so on. But I can not solve (explain) the puzzle when my enzymes migrate at about 40 kDa position in native-PAGE, gelatin zymography instead of estimated 20-25 kDa position. I could explain them as dimers but when I checked the MW by gel filtration (HPLC) I had become really puzzled. They eluted as monomer (22 kDa). I have also used bovine trypsin as control, which didn't show any anomalities. I am really in bad need of explanation. Please be generous to help me out!

#2 anonymous

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Posted 05 September 2001 - 09:00 PM

Dear Ashan,

Though I am not an expert, I have heard of instances where proteins do not migrate according to theirestimate size on small gels (10 cm). I believe that a larger gel apparatus with a longer run time would give better resolution. However, if bovine trypsin migrates as expected, then I am at a loss. One other idea would be to try a different gel system. Novex/Invitrogen has a large selection of gels designed for smaller proteins. I think you might have luck with tricine gels. Good luck!

Naser


#3 anonymous

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Posted 06 September 2001 - 09:00 PM

Your protein is a dimer. The gel filtration sizing must be done in the presence of 5 mM beta-mercaptoethanol and 50 mM magnesium chloride to preserve the dimeric conformation. Keep the pH at 7.6, so the buffer must be Tris.

Good Luck

Nikkita[url="http://www.dstf.bigstep.com"][url]http://www.dstf.bigstep.com[/url][/url]





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