I am trying to plan an pulldown assay to find some prey protein for my bait protein.
I am interested in using the flag tag because it is small and doesn't disturb structure and doesn't require additional steps to tag your protein like would be necessary with biotinylation.
Plus apparently, the bait-prey complex can be eluted with Flag peptide.
So that is very interesting to me.
However, I haven't been able to figure out how strong Flag-Tag binding is to Anti-flag antibody?
Also, does anyone have a simple protocol for Flag-tag pulldown?
Flag Tag = DYKDDDDK
Different monoclonal antibodies have different affinities.
Article Affinity of the monoclonal antibody M1 directed against the ...
Article A high-affinity monoclonal antibody against the FLAG tag use...
Here is one immunoprecipitation protocol:
https://rockland-inc.com/Immunoprecipitation.aspx
Any general protocol for IP should work. Antibody manufacturers may have recommendations for IP and concentrations that they have tested for IP.
In my hands, Flag antibody is OK but not the best. I find HA is cleaner. One thing to consider in IPs with Flag is the amount of DTT in the lysis buffer. DTT weakens the interactions. I now don't add DTT in my lysis buffer if doing IPs with Flags.
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