Question about reducing agent (DTT) - (Jul/07/2010 )

Hi, i was preparing my protein samples earlier today and will load them on the gel tomorrow. While preparing my samples, i cannot recall if i have added the reducing agent (DTT) into my sample buffer used to lyse my cells with. I therefore plan to add DTT to my samples tomorrow (working concentration 50mM), heat them up and then load them on the gel.

My question is: If i have already added DTT previously and add DTT again tomorrow, will there be a negative impact on my results due to too much DTT? Could i 'over denature' my samples?

the samples will probably be able to handle the additional dtt but you should be able to smell if the samples already have dtt (especially at that high concentration).