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Co-immunoprecipitation non-specific binding

co-ip co-immunoprecipitation

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#1 tofuj

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Posted 14 January 2015 - 03:37 AM

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? 



#2 CPRES

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Posted 14 January 2015 - 05:58 AM

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.


So. Now that you have your first ever question on bioforum answered (or not), mail yourself your username and password so you don't forget them, and then come back soon to update us on how it all worked out. That's how you build Karma in science.


#3 tofuj

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Posted 14 January 2015 - 10:47 PM

Thanks for the suggestions CPRES! 







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