The protein I'm working on can function in two forms, monomeric or dimeric and based on that the function changes. I would like to know if my protein behaves as a dimer in certain conditions inside the cell. How can I IP endogenous protein and distinguish between two states?
Hi Sebastian, thank you.
I agree with you, the more I think about it the less optimistic I get. I was thinking of expressing it with a tag, pulling it down, eluting and running native PAGE, banging my head against the wall trying to optimize a western blot transfer conditions and probably still not getting any clear answer....FRET seems like an option, thank you! My protein is also known to form tetramers, but FRET probably won't help to pinpoint that :)
Well with cells you could use more liters of growth media for expression of your protein than you usually do, just so you know that you have a good concentration. Then you lyse the cells, and purify your protein like you do. Then you complex your purified protein with some dye that is sensitive to dielectric changes resulting in a wavelength shift resulting in a new color change upon higher order oligimerization. (For example the dielectric constant within a lipid bilayer is lower than in water because it is more difficult to solvate ions in a lipid. So I would expect the dielectric constant of a dimer to be lower than that of a monomer.)
Then you introduce your protein (probably a monomer at this point) complexed with dye into an appropriate model cell (with only background protein expression) with electroporation of the membrane. Then you could monitor the fluorescence with fluorescence microscopy or flow cytometry. Depending upon the screening of concentrations of protein that you electroporated into the cells you might see the initial population of your protein split into some equilibrium of the monomer and higher order oligomers. Collect your observations.
An issue I see is not electroporating enough protein into the cell (or as much protein as you think would normally be in a cell) and making sure that your protein binds tightly to the dye so that you don't think it will let go of the dye in solution. If you can covalently link the dye to your protein then that is better. Make sure the dye isn't huge so it doesn't inhibit oligimerization.
See you got this! Just need one dye.
Sebastian, over-expression in E. coli easy. It is called William Studier's ZYM-5052 Autoinduction Media for high cell density growth and protein expression.
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