i am attempting amino acid analysis of leaf protein extracts. Upon addition of 5uL OPA reagent to 5ul of extract a black precipitate forms. This precipitate appears to have bound early eluting AA. Has anybody had any experience with this? How can I avoid this?
I prepared the protein extracts by TCA/ acetone extraction followed by phenol precipitation. I resolubilzed the protein precipitates in a rehydration buffer ( 7M urea, 2M thiourea, 4% CHAPS, 20 mM DTT). Samples were hydrolysed with 6N HCl overnight and then dissolved in ddH2O prior to derivatization.
I figure that the issue is probably with the rehydration buffer- perhaps some decomposition product of the thiourea reacting unfavourably with the OPA? I've decided to abandon this technique