coIP troubleshooting

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Feb 17, 2018


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

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Newbie 17425

Scientist, Karolisnka


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relaxin 11 months ago

Can you detect A without IP in your sample anti-A Ab? You need to make sure the antibody is working.

If you do not see the bands with IgG control, then it means the coIP works. If you see the bands with IgG control, it means they are not specific, probably due insufficient washing.
Once you boil the beads with sample buffer, you can spin down the beads and save the supernatant in another tube.

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Newbie 17425 11 months ago

I see the band in sample but not in input. I wonder why did this happen. A master student from the lab repeated it and found the same i.e. there was no band for the input but he got a band for the sample.

That's great. I didn't see any bands in my samples so it has worked.
Can I add more than 25µl of sample buffer to the beads? For example, can I add 100 µl of sample buffer and store it in 4 aliquots?

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relaxin 11 months ago

Are you using the same amount of protein in both input and coIP sample? If yes, then the specificity of the band in the coIP sample may be questionable.

The volume of sample buffer used depends on the amount of target protein in the sample. If you use too large a volume, you may not have enough target to be detected on Western blot.