Hi all,
I was doing protein purification and after that I had to add 2 uL DTT (1mM), but instead, I added 1000 uL DTT (1mM) to my protein solution. I would like to ask you, if my mistake has any bad effect on my protein? I have to run SDS-Page, but I don't know if this amount of DTT would change the results?
best
Amir
You my need to try to see if you can get any positive results.
The best option is certainly to collect new samples, since this amount of DTT would very likely interfere with protein structure / buffer interactions.
If your sample is hard to collect, possibly buffer exchange a few times using a spin column or dialysis. Try to experiment on DTT alone first, since DTT itself may damage the filter in either case.
Good luck!
Your PI will not be happy, DTT is quite expensive. The effect on the enzyme depends on the number of -SH and -S-S- groups, there may be a shift towards -SH. However, chances are that a simple buffer exchange can rescue your sample (desalting column, Amicon cell), especially if exposure time was not too long.
If it is inapplicable to reverse the situation by applying above suggestion and excess DTT is harmfull for your PAGE results you may test to add iodoacetamide to quench excess DTT by alkylation reaction typically made through proteomic applications...I am not sure this is helpful or not but you may give a try at least...
DTT is a reducing agent commonly used to break disulfide bonds in proteins, (aiding in the reduction of cysteine residues). Accordingly, excess DTT may lead to the reduction of non-native disulfide bonds or disruption of the native protein structure. This can affect the protein's stability, activity, and structure.
you can reduce the DTT amount in the buffer through buffer exchange ( size-exclusion column, dialysis, or amicon centrifuge filter). however this doesn't guarantee that you protein remains active.
Good luck
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