DTNB (Ellman's reagent) - (Oct/01/2009 )

Hello everyone,

I need your help. I am trying to do the buffer for MMP assay containing DTNB (5,5'-Dithio-bis(2-nitrobenzoic acid)) dissolved in Hepes 50mM, CaCl2 10mM, and 0,05% BRIJ pH=7,5 but the buffer is yellow, , it looks like 2-nitro-5-thiobenzoic acid were present before the main reaction, I don't know if the problem is pH or purity of components. Maybe someone knows how to make it out, please help.

maybe the dtnb is decomposing.

how old is it?

how was it stored?

The DTNB is a new one , kept in a room temperature as Aldrich advices. Maybe I should change the pH or do a stock in DMSO before??

The DTNB is a new one , kept in a room temperature as Aldrich advices. Maybe I should change the pH or do a stock in DMSO before??

spectrum chemicals recommends storing dtnb refrigerated in a tightly closed bottle.

Yes I have checked if few companies, but some of them advice to keep it in RT,so I don't know what can be wrong. Any other suggestion? Maybe someone has protocol to this buffer?

I know what was the problem, it was CaCl2, when I used different one, now the buffer is OK.