In the instruction of tris-tricine electrophoresis it suggested to fix proteins with 5% glutaraldehyde. In the instruction of silver staining fixation is performed with a solution including 50% methanol and 12% acetic acid and 0.5ml/l of 37% formaldehyde. Is it necessary two do both fixations after tris-tricine electrophoresis.
Probably not, only one should suffice.
However, the wash with methanol/acetic acid prior silver staining also serves the purpose of removing unwanted chemicals (SDS, some ions etc.), thus it is probably advisable to wash at least for a while (although proper washing requires rather longer time).
So it depends on whether you need to fix the proteins with the glutaraldehyde in first place. I always run the SDS-PAGE and then proceed directly to silver staining with the methanol/acetic acid wash/fixation.
As I now see Masood answer - the fixation with glutarldehyde is supposed to happen before or after the electrophoresis?
It is known that aldehydes do react with amines. Therefore, you have to remove Tris and Tricin from a gel before fixation with aldehydes.
Please do it.
Only one fixation step is enough, no doubt...But you can choose methanol/acetic acid because it is very much less aggressive and also reduces the background to staining procedures...
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