help - probe design (Oct/17/2005 )
besides the target probe, how many kinds of control should be designed? competitior probe, non-specific probe, and mutated probe. who can give me a clear conception about those probes?
Btw, when would be the antibody be used?
I am assuming this is also regarding EMSA?
OK, here's the lineup:
1. competitor probe - this is labelled copies of your 'target probe'. you add this at 100X the amount of target probe. So, you have a tube with extract, target probe, competitor probe, and the other constituents of the binding reaction. If your reaction is specific to the sequence of your target probe, then this lane on the gel should have no shifted band. this is because the unlabelled probe(in excess) will compete with the labelled probe in binding of the TF and take up all the TF in the extract.
2. non-specific probe - are you talking about the poly dIdC? this is non-specific bits of nucleotides; it is not labelled and you add it to every tube. this should occupy any random-sequence-DNA binding proteins present in your extract. this is another way to obtain binding specificity with your target.
3. mutant probe - this is very close in sequence to your 'target probe'. you add a couple-bp mutation in the binding site. this should abolish the shifted band. this is another way to show specificity of your TF binding to your target sequence. If you knock out the binding site and the shift is still present, then your TF (or whatever binding protein is present) is not binding to your specific target site.
4. consensus probe - this is not necessary, but it is a good control. get a probe of the consensus binding site for your TF of interest. you can see if overall activation of that TF machinery is induced by your treatment condition, even if your specific promoter does not seem to evince binding
capisce? does this help?
OH, hey, about the antibody...you add an antibody to the TF that you suspect is binding to your target. then, if it's the TF you think it is, you will see a band that is 'supershifted'..or even higher MW than the original shift. this is another way to prove specificity.
> Do you mean UN-labelled probe?? As a competitor probe? Because then it would make sense that there's no supershifted band...
And please, if possible, have a look at the attached files taken from a Promega protocol. I don't understand what the suggested positive and negative controls are for. And if they correlate with any of the controls you suggested.. Maybe it's obvious, but I just don't understand..
And: I want to do an NF-kappaB EMSA. Are there any controls that one should be doing? I can't find anything specific in publications. Will it be sufficient to use unlabelled probe that is very similar in sequence to the NFkappaB oligo?
Thanks a MILLION!