Hi, Praveen, I agree with previous people, 2% will definitely destroying all your protein complexes. You have to use 0.2% Igepal. Moreover, I would suggest to you to use instead of Co-IP PLA assay. Co-IP results were strongly dependent form conditions you have used (lysis buffer composition) which significantly changes protein interactions itself. Finally, Co-IP give you only a probability that proteins can interact under your given conditions. But it did not mean that same proteins can interact in the cells, in the other environmental conditions. So, try first co-localization and thereafter PLA. In this case you will be 100% sure of your results! Good luck!

I think that 2% Triton X100 is much to high. For IHC I use 0.3 % to permeablize the cells. Under this conditions most of the proteins could be stained and are well localized. Therefore I suggest to use a lower Triton X100 solution. Or another detergent like Tween 20? What is in general your IP-protocol. Do you use magnetic beads?

2% seems rather too much for me. The protocols we used had betwen 0,5% (lysis buffer) und 0,1% Triton (wash buffer). If you use too much detergent, you will loose your specific sample.

yeh, it`s to much. maximum, we can used between 0.1% - 0.5% , but its better to use tween 20 than triton X100.

Hi, Praveen, I agree with previous people, 2% will definitely destroying all your protein complexes. You have to use 0.2% Igepal. Moreover, I would suggest to you to use instead of Co-IP PLA assay. Co-IP results were strongly dependent form conditions you have used (lysis buffer composition) which significantly changes protein interactions itself. Finally, Co-IP give you only a probability that proteins can interact under your given conditions. But it did not mean that same proteins can interact in the cells, in the other environmental conditions. So, try first co-localization and thereafter PLA. In this case you will be 100% sure of your results! Good luck!

Hi all,

Thanks for those suggestions. I have used NP-40, Tween-20 and Triton-X 100

at varied concentrations like 0.1%, 0.5%, 1%. 1.5% and 2%. But my interacting protein of choice is binding to the beads even at 1.5% with all the detergents. At 2% only I don't see the binding to beads. So, I am not sure at this high % of detergent whether the protein interaction is stable or not !?? Anybody has idea how to avoid this problem.

In our experience, rinsing several times with high volume in PBS-complete could be enough to get rid of inespecific interactions

Hi I would suggest you that since you have already found that in 1.5% Triton-X-100 (which is very high as it has already been mentioned by others) you are getting your protein of interest in the complex then use that concentration of Triton-X-100 and increase the salt concentration 200 to 500mM in the wash buffer..it works for me.. all the best :)