Gel Supershift - to DTT, or not to DTT? (Mar/24/2005 )
Hello!
I have been running gel shift assays for some time now and have fairly recently began to run supershift assays (I have done about 6 supershift assays). I have not been experiencing any difficulties, but I found a puzzling reference and I am wondering if anyone else has any ideas / explanations / viewpoints.
I found a reference when searching for protocols that says you should never, ever, ever add DTT to supershift reactions because DTT eats antibodies and therefore the supershift will not work.
I am not finding this to be true...my binding buffer contains small amounts of DTT to reduce the proteins in the extract. For the most part my supershifts have been working very well...so why is DTT supposed to be bad?
Does anyone have any comments or experiences to relate regarding this topic? I see the supershift during my assay, but maybe my bands would be stronger, or I would have to use less antibody, or perhaps it would make no difference at all if I cut the DTT...my system works and so I am not going to change it; I was just curious if anyone had any empirical evidence regarding this claim about DTT?
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I have been running gel shift assays for some time now and have fairly recently began to run supershift assays (I have done about 6 supershift assays). I have not been experiencing any difficulties, but I found a puzzling reference and I am wondering if anyone else has any ideas / explanations / viewpoints.
I found a reference when searching for protocols that says you should never, ever, ever add DTT to supershift reactions because DTT eats antibodies and therefore the supershift will not work.
I am not finding this to be true...my binding buffer contains small amounts of DTT to reduce the proteins in the extract. For the most part my supershifts have been working very well...so why is DTT supposed to be bad?
Does anyone have any comments or experiences to relate regarding this topic? I see the supershift during my assay, but maybe my bands would be stronger, or I would have to use less antibody, or perhaps it would make no difference at all if I cut the DTT...my system works and so I am not going to change it; I was just curious if anyone had any empirical evidence regarding this claim about DTT?
:blink:
Hello,
I have done the EMSAs and supershift assays for nearly one year. From the begining, I used Pierce biotin-labeling EMSA kit then I shifted the labeling system to isotope.
To my knowledge, DTT is a strong reductive reagent which will split the inter-chain disulfide bonds of the antibody when the concentration of the DTT you have given is high. Otherwise, I think low concentration of DTT will not affect the supershift experiment. Can you tell me what the DTT concentration that you used? In my case, I used the same binding buffer,which contained 0.5mM DTT, in EMSAs and supershift assays, the bendshifts in EMSA were quite sharp; however, in supershift assay, bends were not clear. So, maybe I should change the binding buffer in supershift assays.
cheer