[SmallRodent]

Hi, everyone! I hope I'm in the most relevant folder of the forum!

Hi, everyone! I am trying to make lysis buffer and when I add the DTT the buffer turns yellow. It's done this twice now. Is it possible that my DTT is bad? There's no way our bottle is more than 4 years old (kept in powder form in a -20 automatic defrost freezer). The only things I had added so far were 1M NaF, 0.5M EGTA, 0.1M Na3VO4, and about 800ml of dd H20. Any idea what's causing the yellowing? Which chemical is most likely to be bad? Thanks!!

Cynthia

[LVJ]

I believe vanadium(V) compounds are yellow. Remember to adjust the pH of the buffer, as vanadate will precipitate at lower pH-values.
Instead of DTT, which is air- and temperature-sensitive, try TCEP-HCl. It's way more resistant to the surroundings and will still reduce disulfides, and the reduction is irreversible and more efficient (as far as I remember).

[SmallRodent]

Hi, LVJ! You are right, the Na3VO4 should have been pH'd to 10 and then boiled until clear. I did not realize this. I just did this and it *still* turned yellow when I added the DTT. Am I supposed to maybe just go ahead and make up the lysis buffer and when it's all done boil it to get rid of the yellow?