SDS-PAGE for A-beta proteins from cell lysate - (Apr/01/2006 )

Hello friends

I am a new guy in working of the field of Proteomics. I am trying hard to review back all stuffs learned from undergrad quite now. My research here involves to detect A-beta 42 protein from PC12 cell line.

Anybody can recommend which percentage of PAGE gel (acrylamide) I should use, in order to separate well out the A-beta 42 protein (which I believe its M.W. is about 4.0kD, from a journal which used Tris-Tricine gel: 10%). Thanks.

Alan

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follow the article's recommendation. i used the same gel for a-beta 1-40.

if you want to fragment the peptide then you can go to a 16.5% or a 10-20% gradient tris-tricine gel.

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Hello again

Thanks for answering me the question.

Is there any difference between using SDS-PAGE gel (discontinuous gel system with Tris-HCl in sample buffer) and Tris-Tricine gel?

Alan

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Hello all again

How about if I choose using 18% SDS PAGE gel to separate my ~4.0kDa protein (A-beta)? Is it possible? As I see the manual of my rainbow protein marker, that this size can be separated, according to the gel image in the manual.

Thanks

Alan

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the difference is that the commonly used version of sds-page uses tris-glycine for the running buffer (method of laemmli). the sample buffer can be the same for both methods (i use the same sample buffer).

i have tried using high percentage gels for low molecular weight peptides and have found that the bands are broad and fuzzy. the tricine helps to sharpen the bands. there is a method that uses urea to help sharpen the bands (Swank and Munkries, i forget the exact reference), but i found that the tris-tricine method works better for the peptides with which i was working.

good luck

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Many thanks!

I think I would try Tricine-SDS gel to run by proteins. However, anyone can tell me if I can use the same gel apparatus (Hoefer), which is usually running discontinuous glycine-SDS PAGE, in this situation? I know the buffer used will be different.

Alan

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the gel is basically the same, just a different buffer system and %age acrylamide. you can use the same plates, spacers, apparatus, etc. you may, however, want to rinse the buffer compartments of the apparatus prior to use.

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