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"weber & osborne" and neville's SDS-PAGE method - protocol please (Dec/17/2009 )

Hi

could any one please provide me with protocol for neville's SDS-PAGE method and weber & osborne SDS-PAGE method?? heard that some times they give sharper bands than laemmli system (this is what we are using in our lab..so curious to give it a try...

thanks a lot

-rick112-

here are the original jbc papers.

i have two versions of weber and osborn formulations, one using a phosphate buffer, as in the paper, and the other using a tris buffer.

we routinely use neville, but, we use a modification. we use a 5-15% acrylamide gradient (it showed better resolution of our proteins than the equivalent laemmli gradient).

if you want, i can post the formulations as we use them (i'll have to type them up, first) but you should be able to get the formulations from the papers.

by the way, sorry it took so long to get this to you, rick112, i was briefly sidetracked.
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-mdfenko-

thanks for the protocol

will give it a try next week..and let all of you know the results...(hope they trun out 2 be good... :lol: )

-rick112-

hi

i was thinking of running the "weber and osborn" SDS-PAGE gel based on above mentioned article...there is no mention of stacking in the article, in case i want to use "Stacking" for my gel could anyone tell me what 'gel buffer' i have to use.
is it the same buffer as separating only with ph difference????

mdfenko...your help will be highly useful

-rick112-

when we ran weber and osborn gels, we ran cylindrical (tube) gels. we did not use stacking gel. with tube gels we normally limited the height of sample load to keep the bands as sharp as possible.

you could design a stacking gel very easily. make the gel ~4% and pH somewhere from 6.1-6.8 using sodium phosphate buffer.

-mdfenko-