This was a question in my protein science final, the only question I missed. Any suggestions? All my professor will tell me is i should run SDS page as first step. I wrote Cryo-EM, which is apparently wrong.
Yes, SDS-page is simplest way provided that they are Disulfide bonded. Like you will run 2 samples : One with normal gel and one on reducing SDS-PAGE. Under reducing page, they will run as a monomer/single band, whereas on other they will run as two bands. You can also do mass-spec on bands to see if peptides that you obtain from the bands are same or not. Another thing is to try native blue gel. Analytical centrifuge will give an idea of exact mass though.
SDS-PAGE would be a simple method to separate and visualize the the monomers. In reducing and denaturing conditions you should see only the monomers. If you see one band at the expected monomer size, then you confirm that the subunits are the same.
But if the two homodimers are run on SDS-Page, they will just show up as a single band no? In theory, you could have two identical size proteins (or very close in size), that would show only one band. How can i prove that both subunits are the same?
Yes, but dimer band will be thicker than the monomeric band. As I said this page experiment is just a face value.
If you want to know more :
1)
If you digest bands and perform mass spec the monomer will give various peak at several M/Z, and in general the dimer will also give several peaks with the same m/z as the monomer which you saw but new peaks will appear at m/z values in between the monomer peaks. You will have peptide match, and you are done - this will provide you very solid evidence.
2)
Another thing which you can do is run analytical size exclusion chromatography, and pull all monomeric fractions together and again concentrate them to the original concentration and re-run that sample on the same column to find out the appearance of dimeric species.
3)
You can do analytical ultracentrifuge to determine dimerization state of your protein. This is also standard biophysical technique.
4)
If you make differentially tagged protein ( Like coexpress Strep and HA-tagged protein ) then detection of HA immunoreactivity in fractions immunoprecipitated with the antistrp ab will provide a reasonable evidence of intermolecular interactions. In the nutshell- there are several things which you can do for this. You just have to think about the facility that is available for you.
Two dimensional gel electrophoresis will separate the subunits on the basis of size and isoelectric point. Even a single charged side chain change could be detected.
Intact protein mass spectrometry by electrospray ionization will show the exact molecular weights of the two subunits, if they are not almost exactly the same and the difference is not too small. It has better resolution than SDS-PAGE.
An x-ray crystal structure would be definitive. CryoEM is now capable of giving atomic resolution structures of proteins, but it is better suited for large complexes than small proteins, I think.
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