Hello,
So, first I perform immunoprecipitation. The eluate is then subjected to immunoblot analysis using an antibody against a related protein (equal size) and gives a band.
How sure am I the eluate is pure ? I've tested the IP eluate on western blot with antibodies against random proteins (for example Actin), and this does not give a signal.
Thanks a lot
Dear colleague:
What do you mean with a "pure elute"?
The elutes from an IP are composed by a lot of proteins. The problem is that you are disrupting all internal membranes and maybe you are creating artifitial interaction between proteins located in diferent compartments.
On the other hand, if you want to validate your result, you must perform the experiment precipitating your second protein, I mean, if you performed your IP against A and found B, when you perform an IP against B, you must to find A). Additionally, you can do a colocalization analisis for your proteins, in order to determine if both proteins are in the same compartment.
Good luck!!!!!!!!
Dear,
I assume that when you perform immunoprecipitation using an antibody against protein A, the final eluate should only contain A? Or am I wrong?
I've also performed IP against B and had a signal with A.
The problem is that both proteins are similar in size, so I can not distinguish on WB.
Lastly, I performed IP on a lysate and I'm pretty sure both proteins are present in the cytoplasm.
Thanks a lot for the very fast answer!
Dear Colleague:
When you perform an immunoprecipitation, the final product is always a big immunocomplex, and depending on your experimental conditions (ionic force, detergent) is de number of proteins obtained. If you want to check how many proteins are precipitating, you can just make a SDS-PAGE and stain with amido-black (even with coomassie blue) to see aproximately how many proteins you have in the complex. Moreover, you can perform a mass spectrum analisys of your elute in order to identify all your proteins.
Good luck!!!!
You got the purpose solved by getting a band for the complexed protein and have a control where non specific protein doesn't gives the signal. So, what is your question? You should be satisfied that IP experiement worked. All the best.
Yes, the IP complex can be too large and contain many factors/proteins and hence there can be lots of non-specific interactions, only with design of other conformational one-one study, one could come to conclusion that what are the interacting partners. Co-IP gives you primary/raw understanding of which factors interact with each other. All the best.
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