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Co-immunoprecipitation non-specific binding - (Jan/14/2015 )

Hi everyone!
I have just learned to do co-ip and I have encountered some problems. I transfected HEK 293T cells with FLAG-protein and HA-protein and performed an anti-FLAG IP and my negative control (HA-protein only) keeps getting pulled down in the IP, I believe this is due to non-specific binding to the beads. As a result I cannot get a conclusive answer as to whether my proteins interact sad.png sad.png

I am considering washing the lysates with increasing concentration of salt in the wash buffer. 

Any other suggestions as to how I can fix this problem? 


Mainly depends upon the stringency of your wash buffer:


- Use more stringent buffers than what you are using now, adding salt would do it, but also adding more detergent can do the trick. (1 M NaCl or  0.5 M LiCl, 1% Tween 20 or 0.3% SDS etc)


Here are some more ways to reduce the non-specific binding:


- Increase the number of washes.

- Increase the duration of incubation in wash buffer.

- Wash at closer to RT if doing in ice-cold buffer, if it is okay with your protein.

- Pre-clear the lysate with the beads (before adding the antibody).

- Pre-block/pre-adsorb the beads with BSA.

- Decrease antibody concentration, perhaps you are using too much of it.

- Decrease number of cells per lysate per IP, too much of over-expressed protein will do it.

- Change the antibody, some antibody batches are more sticky than others (affinity purified or not, monoclonal vs polyclonal, etc)

- Change the antibody, some antibodies are contaminated with other antibody (sloppy colleagues)

- Think, think. Anything that can go wrong will.


Thanks for the suggestions CPRES!