I have to analyze a protein whose pI=8.8. I need to check the difference in electrophoretic motility with and without a ligand; I was wondering about the high pI, if someone know a good running/gel buffer to meet my purpose. All my previous attempts with a Glycine pH 9.6 running/buffer were unsuccessful due to the spreading of the sample as detected by Coomassie colouring.
Thanks in advance.
Can't be much of of a help here. But as a suggestion have you tried running a native gel with the anode reversed?
Dear Fulvio,
I think that you can find a lot of suggestions in this attached paper.
regards.
Francesco
Blue native is kinda tricky I guess (I tried it...). You can also check for clear native electrophoresis from Wittig I, Karas M, Schägger H. (type "High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes" in google and you'll find the article), which should work. Of course, you have to make a long electrophoretic run to make the proteins move...
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