Questions about PAGE - (Aug/28/2006 )
Hi I am pretty new to PAGE and I have several questions I hope some can help answer:
1) What is the difference between continuous and discontinuous native PAGE? As far as I can tell, it is just how you make the gel. I am guessing the stacking gel in discontinuous gives better separation?
2) I am using a Bio-rad Mini Protean 3 Cell. Can I run both continuous / discontinuous gels on it?
3) How much difference will the percent gel make to the separation?
Thanks for helping out!
1) continuous and discontinuous refer to the buffer system. continuous uses the same buffer in the well and in the gel. if there is a stacking gel (not likely but not specifically precluded) it will also use the same buffer.
discontinuous (disc-gel) uses different buffers in the gel and the well (and in the stacking gel, if used).
2) yes, the only difference between them is the buffer system.
3) increasing gel percentage decreases pore size therefore becoming more restrictive.
mdfenko explained it well; one footnote to percentage of separating phase: to choose the right percentage of acrylamide depends on the size or sizes of your protein; to separate polypeptides large than 100 kDa prefer the use of gradient gels or if you like to separate a wide range of sizes such as 15 to 200 - 1000 kDa; in most cases a continuous percentage between 10 and 12% will do for middle sized polypeptides
check a pinned item on role of tris and glycine in page. it explains the functioning and the advantage of using a discontinuous buffer system.