I have been trying to do a co-IP using FLAG tagged and HA tagged proteins. Did another western after transfecting each 1XHA -A protein(55kD) and 3X FLAG-B-protein(69 Kd) in separately and they both express well on their own. But I detect band in only HA tag protein transfected cells after co-IP with FLAG Ab(M2,F1804) and immunoblotting using HA ab(CST).
Hi there,
So you pull down HA tagged protein with anti FLAG antibody? What is the question? Did you run the proper controls?
I make western blotting samples:
1. vector 2. HA protein 3. FLAG protein 4. HA-/FLAG-protein.
I pull down :
i) FLAG tag protein with HA antibody.
ii)HA tag protein with Flag antibody.
i,ii) I expected to detect to HA or FLAG-Tag band in only 4 sample, but i detect to HA or FLAG-Tag band in 2, 4 samples. input data is a no problems. I don't understand how it happened.
Hi again,
So it's either non specific interaction of the protein with beads (you should be able to check this by running the experiment with naked beads only (ie. no antibody)) or the protein tends to precipitate during the process. Do you use antitag conjugated beads, or proteinA/G conjugated beads? If the latter you may try to preclear the protein sample before running the experiment in order to get rid of non specific interaction. Do you saturate your beads with BSA before the experiment?
Thank you for your answer. I used protein A or G conjugated beads (from
GE Healthcare, 17-0618-01 and 17-0618-05 ). I think control samples also appear to band if bead has problems, but I did not detect any band in control sample. I attached to my data, it easy for you to understand.
Hi again, what control do you mean? The true control of the experiment is to run the IP protocol without adding anti-tag antibody.
Hi,
I am also facing and struggling the same problem.
It seems that M2 antibody could recognize HA-tagged proteins in co-IP assay even I elevated the concentration of NaCl in washing buffer from 150 mM to 300 mM (I titrated the conc. of NaCl including 150, 200, 250, and 300 mM).
I agree with Dominique Liger to run the protocol without adding anti-tag antibodies.
However, it could not rule out the possibility of non-specific binding from anti-tag antibodies.
Same problem. I haven't troubleshoot much yet, but I see HA tagged protein pulled by anti-Flag antibody, AND OPPOSITE: Flag tagged protein pulled by anti-HA tag antibody. The lane with normal IgG control antibody is empty, so that's not nonspecific binding to the beads.
Flag AB was M2. HA tag AB was from abm. On a regular Western blot with input lysates they don't cross recognize the tags.
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