If you are using magnetic beads, you don't need to do centrifugation. it would be separated by a magnet only. In the case of agarose beads, you would need centrifugation.
hello! thank you very much for posting these explanations on youtube. just watched the long term potentiation (LTP) and this one about CO-IP and it helped me a lot! just subscribed :)
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Can you please explain how beta actin will be present in the lysates when we only pulled our protein of interest with the beads, all other proteins will be in the supernatant which we will discard or not use for this particular gel. We will run only those proteins which are in pellet and i think beta actin wont be presnt in pellet?
Thanks a lot for the complement. Unfortunately I am not able to reach a big audience somehow...please help me by sharing my channel link with your friends.
If we dont know which protein is interacted with Protein A then how we can use the antibody against the unknown protein. In this case we can only go for sequencing the SDS PAGE bands interacted with Protein A
4:53 how will you get any B-actin band from an immunoprecipitated sample? if b-actin is present this simply means that b-actin is also interacting with the protein A, which is WRONG!
Use IgG heavy chain as a loading control or simply immunoblot [WB] the precipitating protein (here A) to observe that protein A is present in equal amount in all the lanes.
The difference between Pull-down and CoIp is that pull-down using solution from eluted beads, and CoIp uses the precipitate after centrifuge. Then both of them go to SDS-PAGE, is that right?
Co-immunoprecipitation (Co-IP) is a popular technique to identify physiologically relevant protein-protein interactions by using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein.
the target was B. thats why the antibody spotted just B in the western blot. A is there but the antibody wasn't directed against it. a positive band for B (considering that A was pulled down initially) shows there is interaction between A and B
Amazing explanation, however, isn't this an in-vitro technique? Literally everything is happening outside of the body, in a test tube etc. Thanks, nonetheless.
When you are doing from a cell lysate or tissue homogenate then it’s trying to detect the interactions in vivo, while you are doing with purified protein in test tube it’s in vitro....
The interaction between proteins could be either in vivo or in vitro but a technique is never in vitro / invivo.....I hope this answers your doubt.....
QuickQuestion You are studying 3 proteins (A, B and C). You hypothesize that A binds to B (forming a complex AB) but not C. From the scientific literature you know that B does not interact with C. 1) You are able to obtain 100% pure preparations of proteins A, B and C. Which experimental technique would you use to prove/disprove your hypothesis? Describe the experiment and the expected results
