Gradient Gel Electrophoresis - (Aug/15/2013 )

I am preparing 4% to 13% gradient gel for performing BNPAGE. According to the protocol i am following the 4% gel is poured first followed by 13% gel which contains glycerol. This is unlike to the gradient that i use to prepare in SDS PAGE in which high percentage gel is poured first. I wanted to know that is this method of pouring the low percentage gel first replaced by high percentage gel is correct or not? Has anyone of you tried this way or not

Thanks

The conventional wisdom for SDS-PAGE where the separation is largely on size is to pour the highest % first and lowest last as the lowest separates the largest proteins.  However, I don't know how this works for native gels, though I suspect that there should be a size component in there too.

it depends on whether you pour the gel from the top or bottom.

if from the top then you pour high concentration first.

if from the bottom then you pour light solution first.

the protocol that you are following probably uses the bio-rad (or hoeffer) gel casting apparatus where gradients are poured through the bottom and followed by glycerol to clear the tubing and ensure all of the acrylamide is between the plates.

I am encountring another problem in gradient native gel. The gradient gel is prepared perfectly indicated by the marker bands which are very appropriate in proper lane. The problem lies in the sample lane which from the top are perfect but from the bottom they are some what squeezed what might be the reason behid this as i have some bands at te lower end.