Troubleshooting Co-IP?

21 December 2023 0 9K Report

Hi! I am new to doing Co-IP's and needed help troubleshooting.

In short, I have been trying to compare the interaction between Protein A & B and mutated Protein A (mut-A) and B. I know from a previous successful Co-IP that A & B interact. I've been trying to replicate this with no success.

I used a HEK293T culture for transfection.

I used sonication to disrupt the cells - at a fairly low frequency. I sonicated them on ice 4 seconds on, 6 seconds off

My lysis buffer has PBS, 5mM EDTA and protease inhibitor

I used agarose protein G beads for the Co-IP steps

The one major difference was I did not use RIPA buffer to wash my beads while trying to repeat my experiment because of a suggestion from another lab member

I loaded 20uL protein for my gels

My input lanes expressed both protein A & B, but the actual Co-IP lanes only express protein A

I'm not quite sure which step might have disrupted the interaction between A & B. Any suggestions on this would be really helpful! Please let me know if I can provide more info

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Science method

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