I used DTT in protein extraction for WB, but unfortunately it was stored at RT when in the label the storage temperature was 2-6°C. What are the consequences? What is DTT's role in protein extraction before WB? I got only one band after electrophoresis, do you think it's DTT's fault?
Now I have put DTT in the fridge for next extractions but I guess that is too late
Thanks for your help
If DTT is kept at room temperature it will degrade/oxidize. The main role of DTT is to keep proteins in a reduced state. Yes it would likely be too late. If the DTT was left out for more than 72 hours it is definitely too late. Also it is better to store DTT at -20C for long term storage.
Joshua Beytebiere Thanks! So it's about the proteins' state and the disulfide bonds. This means that the reason I got only one band could be another one?
Dear Martina
As Joshua told you the role of DTT is reduce the S-S bonds. It is a strong reducing agent, therefore it react with the oxigen of air and it tend to be oxidative in short time at RT, therefore it is preferable store at -20°C the DTT solutions and once the solution is oxidated you have to prepare a new solutions.
Did you got a single band in SDS-page o WB?
If this happen in SDS-page i suspect that the fact that DTT was oxidated reduce the efficiency of your lysis and you extracted only a fraction of the protein and probably the band that you see is the most soluble with out DTT and the most abundant. But it is just an hypothesis.
Ciao
Manuele
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