DTT or mercaptoethanol - (Dec/11/2007 )
in wikipedia, it wrote: the reducing power of DTT is limited to pH values above 7, since only the negatively charged thiolate form -S- is reaction, the pKa of thiol groups is typically 8.3.
since we are using the buffer around 6, so it is bad to use DTT as the reducing agent to keep the Cys intact?
how about mercaptoethanol? it does not say anything about the effect of pH on it. So is it better to use 2-ME in the buffer?
Thank you.
2-ME will be even worse. Dont forget about it is dose dependent as well.
As much as I like Wikipedia, I'd avise you against using it to answer such questions. I'm not saying that what's written there is wrong, it's probably ok, but you can't be sure. Try to check that on some articles/books/protocols or with someone who knows about the subject. I'm sure you're not the first one to use that buffer so you might be able to get some info on the matter.
Yes, Ambrosio is correct. It could be just one man/woman's opinion until it gets corrected by someone.
But on this issue it shoudl be true to state that reducing power is less at an acidic pH for DTT, it makes sense to me from chemical point of view.
I once had a first hand experience with 2-ME at acidic condition, it took me forever to get my product reduced. I suspected a pH effect, but did not paid much attention until now.
But like usual, you can get around it with more DTT.
how to pose DTT solution? I always dissolve the DTT in 20mM NH4HCO3.
how about IAA?
how about IAA?
we make our dtt stock in dw.
iaa we only make small amounts when we need it and make it in either buffer or dw, depending on what we will do with it.