I would like to know if anybody has ever tried performing co-immunoprecipitation using a mix of two or more antibodies against a partcular protein (I mean not separately, but together in one set itself!). Would it be a good idea to do like this or its objectionable? Your opinion, suggestion and or any suitable reference will be of great help.
No problem. Have done so myself. Just make sure you have enough agarose beads for binding. Expect a fat IgG band!
Thanks Prem. Is it acceptable by the reviewer? Can you send me the reference where if you have published such Co-IP data?
Hello Prem, if you are sharing it with Ashraf via email just add me too :) or please add your paper link in this discussion. Thanks!
Wow, I have never heard of that and would like to have the reference too.
Mixing monoclonal antibodies against a single protein is little different to using a polyclonal mAb. I would say that this is perfectly acceptable and is a great way to increase your pull down without the potential off-target effects that some pAbs have. Remember - it all depends on the mAb specificity.
Yes, we do it routinely for some targets that don't ChIP well with a single antibody (be it monoclonal or polyclonal). As long as you use an appropriate capture method (protein A/G have different affinities for certain antibody species and isotypes), this should work fine. Like Andrew said, there is nothing objectionable in principle, since polyclonals hit multiple epitopes at one time.
Just remember that the more epitopes are recognized by your Abs, the more likely that the Abs will displace any interacting proteins. Ab/protein interactions are among the most high affinity and will disrupt other protein-protein interactions that are weaker in a co-IP if the Ab binding site is near or at the site of protein-protein interaction. This is why C or N-terminal epitope tags are so useful; they are unlikely to disrupt other protein-protein interactions.
I do it sometimes as well ... be careful though with the data interpretation as you need to be sure you are not looking at differences in binding between IPs simply because one of the Abs in your pool doesn't recognise your protein (e.g when using deletion constructs)
I actually have to precipitate potential interaction partners of my protein of interest without any over-expression approach. I already have a positive data using over-expression approach which I need to confirm in physiological condition at basal level. I tried single antibodies but not completely successful yet so thought of trying of using multiple antibodies, but was not sure if its acceptable. Comments and suggestions here are really useful and I thank you all for your responses. I think I will try it out now!
Any reference or article would really boost my confidence though :D
Using two polyclonals increases your chances for variability between ab lots. Ideally get your IP working with a single monoclonal directed against N- or C- term for consistency.
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