Acetyl coenzyme A and dialysis - Removing AcCoA and dialysis problem (Jul/21/2005 )

Hello,

I am an ignorant organic chemist who's trying to do some biochemistry...and so I need suggestion and advise….

It seems that enough of Acetyl Coenzyme A is produced in the culture to interfere after extraction in my enzymatic assays. So in order to diminish the concentration of AcetylCoA , I tried to dialyse the crude protein using the same buffer solution. But if I let the dialysis going over 2h, the protein either precipitate or I am loosing complete activity (and I made sure to wash out the membrane with milliQ water).
The protein seemed to loose a lot of its activity after a partial purification on a dimethylamino cellulose purification. And I encounter the same problem of the dialysis of the purified protein. Is it possible that NaCl interfere in the enzymatic assay?

Does anyone have an iea on what is going on with the dialysis? Or have any suggestion on how to remove the AcetylCoA?

I surely will appreciate any suggestion. Thanks a lot…

My immediate impression - maybe your dialysis pore size is too large? What's the size of the active protein species, vs. the molecular weight cutoff value of your dialysis membrane? If they're too close, you might be dialysing your active species out along with AcCoA.

If you suspect a buffer component (like NaCl) is responsible for the interference with the asay, it's easy enough to test for that - just do a bunch of smaller dialyses varying that component's concentration, and test activity afterwards.

hey thanks.

I used spectrapor 10K and my proteine is about 50KDa and probably hydrophobic. So now I think that it might stick on the bag or I read somewhere that some proteine tend to precipitate in the dialysis bag when they are to concentrate...

But I will try the activity vs NaCl concentration assays

Thanks again