Protein stuck in the origin of resolving gel - (May/26/2015 )

what is the ratio of acrylamide to bisacrylamide? standard for protein gels is 37.5:1 (sometimes 29:1).

if you are using a greater crosslinker ratio (eg 19:1) then the gel will be more restrictive and will require even lower concentrations to allow high molecular weight proteins to migrate farther.

It is 37.5:1 (2.6%C). I have no problem extracting the problem, or temperature, reducing agent, transfer,  it is just that my 260kDa protein is stuck on top as well as my 250kDa protein marker (at least a faint band). I really don't know what else to try. Any suggestions would be helpful at this point.

we look at a wide range of protein sizes ( we used to work with myosins), so we routinely use a 5-15% acrylamide gradient with a 4.5% stack.

if your only protein of interest is > 200 kDa then you may want to try a 5% gel with a 4 or 4.5% stack. be careful, the gel will be fragile and difficult to handle.