Fixation on Acrylamide - Fixing a Peptide on Bio-Rad Tris/Tricine SDS-PAGE (Sep/18/2007 )

Hi anyone,

I'm having a heck of a time trying to fix low molecular weight proteins to tris/tricine gels - both the Bio-Rad and ones that I pour myself. Basically, i have a marker that goes from 16 down to 2.5 kDa. The 6 and 2.5 kDa markers, and thus any other proteins will not show up by coomassie nor silver stain by conventional fixation, but only with glutaraldehyde (5%). On top of that, it has to be an overnight fixation. If I go for only a few hours...no bands below 8 kDa. This long fixation means that washing with water takes nearly the entire day. The gel never loses the yellow color from the glutaraldehyde. Subsequent staining by Coomassie has significant background or even smudging, especially when washes are not sufficient. My protein is 1.3 kDa, but when I run it on the 16.5% Tris/Tricine Peptide gel (Bio-Rad) its apparent MW ~6-10 kDa. My goal is to ensure fixation by visualizationi with coomassie blue (0.25%). I want to:
1. speed up the process - would wash with Ethanol prior to water improve resolution
2. is there a better fixation method
3. I use GE Healthcare globing MW markers (16.5 down to 2.5 kDa) and I think they are crappy. Is there something better?
4. I used lower % Coomassie recently and have not seen the peptide bands. Perhaps I should not boil the peptide or I should keep Coom at 0.25%?

Thanks for anyone's suggestions.

DOCDTT

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invitrogen mark12 unstained standards may do the trick for you if you only need to go down to 2.5 kDa.

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Thanks.

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when we run tris-tricine-sds (shagger and von jagow) gels we normally silver stain (based on the method of merril, et al). the method we use to stain starts with fixation with methanol-acetic acid (50%-12%) and all the peptides seem to remain in the gel.

you may also be able to use tca for fixation but you will need to wash the gel thoroughly before adding coomassie.

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Thanks. I've tried methanol fixation (even overnight) but i just doesn't work in my hands with these gels. How long do you fix?
How low are your peptides? And, do you have a TCA fixation and wash protocol?

Thanks

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we would fix for 20 minutes. by the way, acetic acid is the fixative, methanol prevents the gel from swelling (and, at 50%, will actually shrink the gel) and removes the sds.

the tca method is one that we rarely used (and it has been a long time). you soak the gel in 10% tca for 20-30 minutes, until the proteins are denatured, then wash out with water until you can't detect tca (we did it pretty much by odor). you have to remove the tca because it will cause the coomassie to precipitate (which may not be such a bad problem if you are using a colloidal stain). the gel will swell so you can add some methanol to the solution and the wash, if you want.

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Thanks a lot. I'll take a look at it. I just did a gel but used only 10 ul (for a 30 ul well) and got better resolution. I think the band diffuses when 10 ul is exceeded. However, that will dictated how much peptide I need to use (10 ug gives the best visualization).

Thanks for the tips.

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