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.
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?
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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.
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 :)
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
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@@animatedbiologywitharpan anytime i search up biology content, this girl "bumbling biochemist" pops up. her stupidly long videos monopolize biology content, I swear, every single topic i search up i see her face.
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
6 in-depth lecture slides couldn't help with what you did in 4 minutes! Thank you so much.
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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.
BEST EXPLANATION I'VE GOTTEN! THANK YOU!
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Great video! Super clear and easy to follow... I also liked your diagrams. Thanks for sharing
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Very good explanation, thank you so much, and the diagrams are so neatly-drawn too! Was so confused about co-IP and IP before this haha. Thank you!
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Lovely diagrams and nice clear explanation!
The best explanation l have found. Thank you
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This was PERFECT! Thank you! Also you have very nice handwriting!
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@@animatedbiologywitharpan absolutely!
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?
YOU SIR ARE GENIUS !
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Check out all my playlist...you would definitely find topics of your interest
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!
very true.
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.
Im a medical student,Thank you so much for this video❤️❤️❤️❤️it helps a lott!
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Do checkout my other playlists
Thanks a lot. your videos are really helpful for young researchers.
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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 :)
Please share among your friends and help me to reach big audiance
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
You would have to go with mass spectrometry though. Sequencing is used on genetic material, not on proteins.
thanks this is short, concise and logical, made it easy to understand
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very helpful video, the only video explains co-IP with western blot!
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a very straightforward video ! thanks a lot !
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Thanks for explanation! It was really helpful
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OH MY GOD THIS IS SO GOOD!!!!!!!!!!!!!!!!!!!! THANK YOU!!!!!!!!!!!!!
Could you please help me by sharing my contents with your friends group/ college group. I put huge efforts in making these videos but unfortunately not a lot of people are watching this.
@@animatedbiologywitharpan anytime i search up biology content, this girl "bumbling biochemist" pops up. her stupidly long videos monopolize biology content, I swear, every single topic i search up i see her face.
Love your hand drawing!
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Explained very clearly thank you!
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thanks a lot! from Argentina!!
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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?
I don't understand why we need a second antibody for protein B! Would it not show anyway on the SDS Page? Or did I miss something?!
Thank you man, it will facilitate my presentation. You are the best :))
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Super useful, thank you !!
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Very nice presentation
Nicely explained explained. Could have been better if the importance of running input in the same gel was also given.
I agree with your suggestion.
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.
Thanks for the very nice video. But I have a question. How can we detect unknown protein bound to the know protein in co immunoprecipitation ?
Atleast you need to have one known protein which you would pull down and look for interactors via mass spec
how do you justify the pH of the buffer to make sure that protein A and B are not dissociated?
So helpful, thanks!
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Do you have this information from a source? If so, which one? Can you send me the link please
You liked my reaction, but you don't answer?
How we select antibody for a protein,
How we can sure that our antibody interact with our protein??
ua-cam.com/video/U76Ll3OuBsU/v-deo.html
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Where is protein A on the blot? How is A not blotted?
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
does this only work for covalent interactions?
No no generally protein protein interactions are non covalent.
actin is used just to make sure the proteins you are testing presence for were loaded correctly on the blot?
Actin is a housekeeping protein so it’s not expected to change
THANKS! NOW I CAN UNDERSTAND A PAPER THAT I HAVE TO READ xD
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It's fantastic thanks!
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good job brah...keep uploading
Thank you, sir :)
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thank you!
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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.
Nope ...you got it wrong..... this method can be used to detect both in vitro and in vivo protein protein interaction....
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.....
@@animatedbiologywitharpan ohhh okay. Yes that clears the confusion. Thankyouu 😊😊
@@animatedbiologywitharpan ive been taught something so different 😂😂😂 and ive a paper in about an hour.
tell me are u from India? what is that accent??? i need to know
Yes I am Indian, sorry if my accent has bothered you.
great explanation
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thank you
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thank you brother!
r u from Presidency? very good keep up the good work bro..👍
I am from TIFR
Sir it is in vitro technique????
Could be used for in vitro or in vivo
What is the purpose of using beta actin in this assay sir?
It's a house keeping control......you can imagine it to be standard scale
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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
Email me at arpanparichha1994@gmail.com. we can interact further
Who teaches better?
A. Your professor majored in biotechnology with 10+ years teaching experience
Or
B. A random indian boi on youtube
It depends on the audiance
so by this way you describe yo identify the complex AB not their interaction as say !!!
good presetation, except some accent, but it's good. thanks.
what is this accent ?
fakeha khan this is Bengali accent....sorry for that
Thanks!!
Welcome!