I am trying to confirm whether my recombinant protein of interest is a multimer. The only technology I have access to is native PAGE. What I know is that the monomer is a 43 kDa cytosolic protein. I suspect it's a tetramer, so expected native size would be 172 kDa.
So far, I tried Novex™ Tris-Glycine 8-15% gel and NativePAGE 8-16% Bis-Tris blue page system from Invitrogen. In both cases, my protein just sits within the well and does not move during the electrophoresis. Even the addition of G-250 to the sample prep, which is meant to empose a negative charge on the protein, did not make a difference.
Can anyone please suggest what else I can try?
What is the isoelectric point of your protein? If it is above ~8.5 then it is not going to enter a standard Tris-glycine gel because of it's positive charge. A suggestion from Bio-rad for using their Tris-glycine gels with native page samples having an isoelectric point above 8 is to reverse the polarity of the electrodes during the electrophoresis, although I have never tried this. It is likely though that by doing this most of the molecular weight ladder markers will either migrate abnormally or not enter the gel.
Blue native page is generally better than standard native page as the coomassie G-250 adds a negative charge, but one problem is that it relies on the binding of the G-250 to the proteins and there may be insufficient binding capacity. Some proteins will bind different amounts of G-250 molecules because of their individual properties, and some may not be able to bind a sufficient number to be given a sufficiently negative charge. How much G-250 did you add to your samples and was this added immediately before loading? You may need to add more G-250.
Also consider the possibility that your protein is highly aggregated, as sometimes happens with overexpressed proteins. Gel filtration chromatography and dynamic light scattering are good ways to test for aggregation.
Samuel Davis Thank you for your reply. I added 0.5ul of 5% G-250 immediately before loading. According to the protocol I could go up to 1ul which I will have to try.
Adam B Shapiro I suspect there could be some aggregation, although I can not visualise any clumping in the solution as such. Do you have any suggestions as to how I can "de-aggregate" without denaturing the protein? Should I be adding detergent to the sample?
Adding some detergent (digitonin, n-dodecyl-β-D-maltoside) could help to facilitate protein entry into the gel.
How long and at which voltage did you run your gel? Can you run it at low voltage overnight? Have you tried to transfer the proteins to a membrane (denaturing conditions) for Western blotting? Even if Coomassie does not show anything directly in the gel, the protein may be in the gel. A problem you could face is that the Coomassie interferes with the antibody binding. If this becomes a problem, you could run a clear native electrophoresis.
If you have a UV-VIS spectrophotometer of Nanodrop available, take a UV spectrum of your protein. If the protein is aggregated, you will see that the expected 280 nm peak is shifted to a shorter wavelength, or there may not even be a peak. This is because of light scattering from aggregated protein particles, which increases in intensity as the wavelength decreases.
Adding a mild, nondenaturing detergent once the protein has already aggregated probably won't work to disaggregate it, but it would be easy enough to try, using migration on native PAGE as the readout.
Gel filtration chromatography gives you a better idea about the size of aggregate. Although you don't see any aggregation in the solution, sometimes it can still form soluble super complexes which might not enter the native PAGE and precipitate at the bottom of the well.
NAtive PAge does not work for protein it works for DNA. If you have to confirm protein of your interest knidly use SDS PAGE
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