Hello all,
I am looking for interacting candidates for my protein and after doing IP followed by mass spec (found twice), I found protein X to be enriched almost 1:1 ratio to my bait protein. However, during validation, I pulled down with M2 sigma mouse flag antibody (my protein is flag-tagged and overexpressed) and blot for protein X antibody which is rabbit polyclonal.
In HEK cells, I got desired results (2 times), specific bands in my IP lane, no bands in different control lane and everything went perfect except the protein is running a bit higher but that could be PTMs . However, when I tried to repeat this CoIP in a mouse cell line (this is the one I am interested in particularly and providing kind of endogeneous environment) .I am getting a very intense band for protein X in mock IP lane also.
I tried 2 hours incubation to overnight incubation with varying beads and all. Still no luck.
Do you have any idea what could be the problem? Is there any chance of cross-reactivity or something?
I am attaching here a snap as well in case it helps.
Thanks.
Predicted molecular weight is 100 kd. the upper band is running 15-20 kd higher i guess!
really hard to say what has happened. have you been using exactly the same conditions for eluting the sample from the flag beads. were the beads for the two experiments from the same batch?
a frequent problem is the elution of immobilized antibody (anti flag in your case) from the beads. these will later show up as immunoreactive bands on the blot. this, is due to a cross-reactivity of many commercial secondary antibodies. but this does not really fit for the things you present.
I would be concerned about cross-reactivity. Can you try an antibody from a second source?
It may also be worth your while to play with the lysis/precipitation buffer, with both higher and lower concentrations of detergents. We dialed back detergent concentrations, which eliminated some false positives.
Eric... I generally use 1% triton-X in my lysis buffer. Would you recommend going higher or putting low amount of sds maybe?
J. Stolz... I also think that it might be denatured anti flag, but why do you think it is not fitting in this case. And the beads were from different batch indeed.
I would try both a higher percentage and a lower percentage. You might be exposing some hydrophobic patches leading to nonphysiological interactions.
If your beads are from a different batch, you may need to repeat the original experiment to make sure that it's not doing the same thing in the other cell type.
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