I am running a native gel for my protein. Its purified and it runs perfectly in a SDS-PAGE. But when I go for a native gel electrophoresis, the protein gets stuck in the stacking gel itself and does not enter the resolving gel. I am running the gel at room temperature.
SDS denaturate proteins and homogenize charges which facilitate migration . Native proteins of course keep their structure and charge. Two factors that may compriomise migration through the gel. Size and possible precipitation are also important.
Do your standards run correctly? If so, precipitation is probably the problem.
Have you tried flipping the voltage? Without knowing more about your protein (e.g. pI) and buffer (pH) it's hard to say if that's the problem, but it could help. Native PAGE separates based on charge, size and shape of protein in the native state.
My protein has a pI of 5.76 (from PROTPARAM) and the buffer in which I have suspended it is Tris-Cl (7.5). I believe precipitation is the problem. I need to check the interaction between my protein and another protein (pI 5.8) so I do not want to add SDS. Michael can you please elaborate for what do you mean by flipping the voltage? Thanks...
My apologies, by flipping the voltage I meant switching/flipping the anode and cathode. However, with your protein and buffer I don't think that's the problem (crucial for basic proteins versus your slightly acidic protein).
If your protein tolerates it, I'd try different buffers farther away from the pI, say Tris-HCl pH 8.5 to see if that helps increase the net negative charge.
I see now that you are trying to test protein:protein interaction. Have you looked into a recombinant co-IP approach? You could leave the affinity tag on one of the proteins, purify the other and then mix them. Co-elution would provide strong support of interaction at that point. Alternatively, gel filtration chromatography (individually and together) would provide orthogonal support as well. Good luck!
Small amounts of urea (2M) sometimes help without denaturating the protein and avoiding precipitation. Post translational modifications like phosphorylation (if the protein is not expressed in procaryotic organism) should be taken into account for PI calculation.
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