Dear Scientific community,
I have a protein (tetramer) which has a NADH bound to it (confirmed from the crystal structure) when it is purified from Bacteria.
Additionally, I have two mutants of these protein (Dimer and monomer) which I am pretty sure does not contain NADH/NAD+ bound to it.
The point is I need to prove this experimentally and I was told to use C18 column for this purpose and perform analytical RP-HPLC.
So my question is about the experimental setup!So how will I denature my protein and what will be my buffer A and Buffer B? Also some suggestions on the specifics of the protein concentration to be used.
My ideal set of samples will be:
1) NADH only (positive control)
2) Protein WT (tetramer)
3) Protein mutant (dimer)
4) Protein mutant (monomer)
Any input will be appreciated!
Thanks,
Nishit