Hi everyone,
I have doubts about oligos used in fluorescence anisotropy for looking at protein-DNA interaction.
Do both oligos need to have FAM at the 5' end? (I guess the positive side of labelling both strands is greater sensitivity, but the negative is greater chance of artifacts.)
Do they need to be HPLC or PAGE purified? Does it make a difference worth the price difference?
Annealing protocols for making the duplex vary substantially in the literature. My oligos will be short (less than 30 bp long). I expect raising to 95C then slowly cooling to RT will work. Anything to the contrary?
Also, would you use the same oligos in EMSA/gel shift assays? If the protein affinity for the duplex is in the nM range, would EMSA work best than fluorescence anisotropy?
Thank you!
I don't think it is necessary to label both oligos. One FAM should give sufficient sensitivity for most purposes. If you decide to label both, make sure the labels are at opposite ends of the duplex so that they don't quench each other.
Care should be taken to make sure that the FAM probe is not the main source of binding affinity. You should do a control experiment to make sure the unlabeled DNA can fully compete off the labeled one.
Your procedure for annealing sounds fine. It's pretty standard.
I think it is worth having the oligos purified by PAGE or HPLC, despite the added cost.
For nM binding affinity, I would expect good results from both EMSA and FP methods.
By the way, if you have the necessary optical filters for it, consider using TAMRA instead of FAM. It is less sensitive to photobleaching.
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