Dear CCP4 community, Sorry for the off-topic subject, but I would really appreciate some suggestions and/or protocols relating to native gel electrophoresis of basic proteins. I have used a general acidic PAGE protocol for my protein, which has a PI of 9.5. Briefly, the protein was loaded onto a native gel (I have tried both the pre-made Biorad gels (7.5% and a gradient gel: 4-15%) and freshly prepared Tris native gels adjusted to pH 6.8) and run in a 1X acetic acid/b-alanine pH 4.5 running buffer. The electrodes were reversed and the gel run on ice for ~ 2hrs at 100V. In all cases, the native protein was unable to enter the gel. Some protein samples incubated with heavy atoms were able to enter the gel (possibly indicating binding) but these samples too had problems entering the gel as the bands were at or just a little bit below the edge of the well. Any suggestions and comments would be most welcome!
Thank you so much in advance for your help, Sincerely, Ming Lye
