Hi Laura,

the pH of your mate's gel is very close to the pI (isoelectric point) of his protein of interest - since proteins lose net charge at their pI, this would explain why they are not entering the gel. The shift you want to observe depends on the charge of the protein/complex, so it is a good idea to allow for charging by using the correct pH.

I would propose to use a buffer system with a pH approximately 1 pH-unit off the pI (any direction). Try to use the same buffer for sample preparation and gel running (sample buffer = running buffer) to avoid precipitation due to abrupt pH changes when the sample gets into contact with the running buffer (sample application into the well). The pI might be crossed shortly during sample application, causing the protein to crash out of solution in the well.

A first idea would be to run the gel in a buffer system having the same pH as your binding buffer, which is 7.5 - for example, the Novex NuPAGE system by Novagen uses such a pH, if  I recall correctly.

Best of luck,

Peter

Thank you Lucia. My protein seems to be stable in binding buffer (I used DSL to check it). I also use 7.7% glycerol in binding buffer. I will check if changing pH of running buffer and then probe/protein ration will help.

my sample and marker stuck in gel what is the problem

please suggest this is second time