[Green_Meh]

I've done a search on here and on Pubmed and I'm coming up with now answers so i was hoping someone could help as this problem has me stumped.

I've got some chemically modified RNA and DNA which has a -S-S-R group on its 5' Phosphate, and I also have some stock primers that have been bought which have 5' Phosphates -S-S-R too (using these as controls).

Now I've check the literature and the only way I can see to test for this is to reduce the S-S down to a thio and use Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid on it. You obviously have to purifier off the reducing agent otherwise that will react with the Ellman's Reagent. Now what should happen and I've done this with DTT dilution series is the absorbance at ~350 should go down and the absorbance at 412 should go in massively. However it's not very sensitive and there is approximately (I'm missing my notes at the moment) no signal difference between 100nM downwards.

The spectra basically looks like unreacted ellmans reagent which doesn't have a peak at 412.

I've been trying this for about a week and I'm out of ideas to test if the reaction has been successful...and it's even more odd that the controls (the pre-bought DNA doesn't react with the ellmans.)

Now I've ordered in some Fluorophores with a thiol which should in theory react with the thiols in the DNA/RNA samples and then after purification any signal seen should be in threw be from bound fluorophores.

In the mean time does anyone have any other way to check for -S-S- or Thiols? Or can directed me to a paper that uses a method?

There is one other test with 2-Nitro-5-thiosulfobenzoic acid which i found in a paper which looked promising from (1986) however you can't seem to buy this anymore....so thats out of the window...

[Babcock_Hall]

Anal Biochem. 1998 Dec 1;265(1):8-14.
Evaluation of methods for the quantitation of cysteines in proteins.
Wright SK, Viola RE.
http://www.ncbi.nlm.nih.gov/pubmed/9866701
Not sure whether or not this will help.