DTNB (Ellman's reagent) slow reaction - (Sep/08/2013 )
Hi all, I need your help.
I'm testing the concentration of reduced sulfhydryls in my thiolated DNA (DNA-SH) solution using Ellman's reagent (DTNB, 5,5'-Dithio-bis(2-nitrobenzoic acid)).
The thing is, from the protocols I've read the reaction should reach completion after 15 min at the longest, but I'm seeing a continual increase in 410nm absorbance past 40 mins. I've tried buffers of 0.1M PBS (pH 7.0) and 0.1M PBS + 1mM EDTA (pH 8.0).
Absorbance readings: 0.000 for blank, 0.006 after 10 min, 0.012 after 40 min.
My sample is ~100uM 5' thiolated DNA oligos ordered from IDT (standard desalting) in 250mM NaCl + 25mM PBS.
Any guesses as to what's going on here?
Thanks a tonne!
I've also seen this and couldn't figure out what was going on. It doesn't make sense because you would expect the opposite effect (you would expect a decrease in absorbance from re-oxidation of the DTNB).
I've found the best way to quantitate thiols with DTNB is to include a standard for comparison where the number of thiols are know. You can use freshly made dithiothreitol (DTT), make a standard curve and compare to your thiolated DNA. It should eliminate the slow change over time issue.