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coIP troubleshooting

coIP immunoprecipitation western blot protein-protein interaction

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#1 Mad Researcher

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Posted 16 February 2018 - 08:40 PM

Hi,
 
Today, I completed my 1st coIP experiment successfully. However, I have a few questions:
I had two proteins of interest (A & B) and I wanted to see if Protein C pulls it down. I found that Protein C pulls down protein B (25 KDa), but not protein A (~70 kDa).
What could be the case for not detecting A? Is it the antibody or there is no/weak interaction. A recent interactome study has shown that A & C interact and have a good interaction.
 
I saw bands in my 2 inputs and 2 samples but I couldn't see any bands in my IgG. Does this mean my coIP was ok? What if bands were detected in the IgG controls?
 
Lastly, once I prepared the beads, I added 25 µl of Sample buffer and I loaded the same to my gels. Is there any way to store the beads with or without sample buffer for longer time so that it can be re-used in case my experiment fails? 
 
Thank you 

Cheers,

Mad Researcher

#2 Trof

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Posted 17 February 2018 - 10:44 AM

You need to check if your A antibody works fine, if it does, and IP also works (it does, if it pulls down other protein) then the complex you are looking at is either not so stable or indirect, which may be affected by IP stingency, or there is no interaction (at set parameters, cell lines).

 

Not sure about your naming of the non-IP lysate and the one from IP, so maybe better post an image?

In the IP lysate, you should be able to detect the protein you used for pull down (i.e. C) in any case. Also abundant IgG used for pull-down causes two nonspecific bands of light and heavy chain in secondary antibody, if it is the same type (i.e. mouse IP Ab in secondary mouse Ab), which are 25kDa and 50kDa in size, so question is if you did detect a specific band for B at all.

 

If you talk about preparing beads and sample buffer, I guess you mean Laemmli added to your IP lysate? That mix can be stored in -20 as other lysates in Laemmli. (though if you have small cell number, it may not be enough for multiple gels, I grow 10 cm dish of HEKs for coIP and use around 90ul of Laemmli for ~20 ul bead-and-some-dead-volume residue)


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