Hello,
I do IP using HA-tagged beads. One of my proteins through which I precipitate complex has HA-tag. I see this protein (with HA-tag) after IP. The signal is strong and bright.
The complex contains several proteins. For each protein I do separate western blot (I have different membranes for different proteins - I do not do re-probing). Proteins without HA-tag are not very abundant in the precipitated complex and I need use Clarity max (Femto) staining to see them. As a result I see as proteins that I try to detect as HA-tagged protein. The signal from HA-tagged protein is weak but it is still crucial for me because one of proteins that I am looking for has size slightly bigger than HA-tagged protein.
I tried to use true blot secondary antibody but it did not helped. I tried to use different primary and secondary antibodies, for example antibody fro HA-tagged protein is produced in mouse and I use anti-rabbit primary and secondary antibody for other protein of my interest but it did not helped.
I do blocking of membrane in 5% milk with tween-20. Primary and secondary antibodies are also diluted in milk. As an option I tried to block membrane and incubate with antibodies in 5% milk with TBS-T and wash it with TBST but it did not helped.
I cannot increase number of washes because I will loose proteins without HA-tag.
Does somebody has an idea how to solve this problem?
Looks like your HA tag has nonspecific interaction with your secondaries. You can try to change your approach and pull down the protein of interest by itself without an HA tag. You can change your plasmid and switch to FLAG or Myc as the tag as well.
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