While doing NATIVE-Gel electrophoresis with electrophoresis buffer containing 25mM Tris, 192 mM glycine, and rest water if the sample being loaded is dissolved in methanol it won't remain in the sample well. Every time it comes out of the well, maybe something to do with the difference in density of electrophoresis buffer and methanol. If the sample being loaded is dissolved in any buffer (as sodium phosphate buffer for example) no such issue occurs and it stacks up properly in the well. For methanol dissolved samples do I have to make any changes in the electrophoresis buffer? Please help
regards
Try including 10% glycerol in your sample buffer to increase its density.
Hi Vivek Chauhan,
Did you treat your protein with a sample buffer contains methanol? I have never heard that before. I've used methanol in the fixed solution for the staining step.
I totally agree with Dr. Adam B Shapiro, glycerol helps pull protein down in the well. And please notice that the protein migration depends on gel pore size (resolving gel percentage), or might be your protein did not complete dissolved yet?
Best luck!
Why are you loading protein in MeOH for NATIVE-PAGE?
You definitely have to add glycerol, but I'm not sure 10% will be enough. This problem is common with nucleic acid purified using EtOH precipitation.
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