I am trying to find out the binding region on protein A for protein B. I have made different truncated clones of protein A with 3HA tag. Amazingly protein B is binding to all of them! Although the truncations are designed intellectually lacking several domain in each. I tried CoIP with FLAG and Myc tag of protein B but the result is same. Any suggestions please? Does a protein can bind to 3HA tag; as only it can pull down all the truncated clones?
Try to remove the tag of one of your proteins before doing the binding assay to see if you will get the same or different results.
Good luck
Hi Uroos, in addition to Aamals suggestion you can also try to test a reference protein (i.e. GFP) with a 3HA tag - if this also binds, you may have a problem with your pull down assay protocol or your protein of interest.
Good Luck!
Hi Uroos,
Not sure if I understand your experimental set up. However, I know for certain that many proteins bind to HA or FLAG tags. This is confirmed by proteomics experiments where interactomes are studied. Regardless of what the bait protein is, we always see a number of proteins identified (they are actually NOT commonly-seen proteins like actin or tubulins) in all the experiments and they differ if HA or FLAG tag is used.
Best,
Hediye.
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