Did anyone do the oligonucleotide pull-down experiment before? My no DNA-probe group still can pull down the target protein using the streptavidin beads. Does that mean I need to do the pre-clear step using the beads before adding everything?
I would not do a pre-clear step if it's your target protein binding non-specifically to your beads. You can try changing your wash buffer (adjusting salt or detergent concentrations) or blocking your beads in BSA before you use them to reduce the non-specific binding. Depending on what your protein is, it may also be worth checking it for motifs that could bind streptavidin.
Theresa Smith
Hi Theresa, thank you so much for your useful information! May I ask how to check if my protein has the streptavidin binding motifs? My protein is p65 (RelA).
I'd pull up the sequence of the protein and then look up what sequences in of biotin bind streptavidin and test them for any alignment. Also if your target protein is overexpressed, it could be good to just make sure that you know the sequence and any tags it may have, if you don't already. I once had an IP repeatedly not work and it turned out that another protein I was expressing had the same tag as the one I was trying to pull-down, but I didn't know it was there.
Theresa Smith
It is really very helpful! Thanks for sharing your experience!!
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