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

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