What is the purpose of adding the DTT into the SDS-loading buffer just before use? Is it wrong to mix together all substances (i.e. with DTT) and then store them at -20 °C for a prolonged time?
If in doubt about a batch of sample buffer where DTT/2ME were previously mixed in, you can always compare it with sample buffer without reducing agent, using a protein whose migration changes noticeably when reduced (e.g. BSA).
This (and links therein) will probably interest you:
http://lclane.net/text/dtt.html
As DTT is a strong reducing agent and also it oxidize over time it should be added before use. Hope it will help u.
It is a sulfhydryl reducing agent which gets oxidized very fast unlike mercaptoethanol and so it should be added just before use as the oxidizing agents present in the medium like peroxides will make it non-functional
I prepare the complete SDS-SB with 2ME (or DTT; I prefer 2ME though) and store in small (1ml) aliquots in -20. I store the in-use aliquots at 4C, and have not faced any difficulty so far! DTT does just fine. The molar fraction of the reductant undergoing oxidation is rather small, or that is what I believe. By the way, Siva, where do you find peroxides in the medium for SDS sample denaturation? It only has some Tris-Cl, SDS and glycerol. The gel has the peroxide, but the SDS-SB does not see it unless it gets into the gel!
If in doubt about a batch of sample buffer where DTT/2ME were previously mixed in, you can always compare it with sample buffer without reducing agent, using a protein whose migration changes noticeably when reduced (e.g. BSA).
This (and links therein) will probably interest you:
http://lclane.net/text/dtt.html
Alejandro, thank you very much for your comment, the link you provided is indeed very interesting and useful
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