Co immuno precipitation

[@novar7739]

6 in-depth lecture slides couldn't help with what you did in 4 minutes! Thank you so much.

[@animatedbiologywitharpan]

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[@biswajitbiswas2960]

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.

[@jacquelinelabovitz4613]

BEST EXPLANATION I'VE GOTTEN! THANK YOU!

[@animatedbiologywitharpan]

glad to know it helped you.....share with friends and help them as well....happy learning

[@sadafbahadori1453]

That was great

[@mubashirjaved63]

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?

[@AAAdeeno]

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!

[@animatedbiologywitharpan]

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[@j2zel]

Great video! Super clear and easy to follow... I also liked your diagrams. Thanks for sharing

[@animatedbiologywitharpan]

Thanks.... good to know that it helped...now share among friends to help them as well

[@CancerSleuth]

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!

[@tsamchoe1638]

very true.

[@CancerSleuth]

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.

[@chunchom.s]

You save me! Really thank you Your video is precious!!

[@animatedbiologywitharpan]

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[@animeloverXinuyasha]

Lovely diagrams and nice clear explanation!

[@ambergupta8639]

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

[@Echodonut]

You would have to go with mass spectrometry though. Sequencing is used on genetic material, not on proteins.

[@CR-uu1rr]

Very useful and excellent explanation. Thanks a lot 👌

[@animatedbiologywitharpan]

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[@fatnmchollandl6505]

The best explanation l have found. Thank you

[@animatedbiologywitharpan]

Fatn McHollandl please share and subscribe to my channel

[@mahatai9904]

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?!

[@allienikole18]

This was PERFECT! Thank you! Also you have very nice handwriting!

[@animatedbiologywitharpan]

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[@allienikole18]

@@animatedbiologywitharpan absolutely!

[@oussamakherbouche6562]

YOU SIR ARE GENIUS !

[@animatedbiologywitharpan]

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[@animatedbiologywitharpan]

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[@naboclare]

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 :)

[@animatedbiologywitharpan]

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[@iyXi0823dqx]

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?

[@johntough7936]

Really nice video! Though I would re-visit some experimental procedure videos and stumbled upon your channel! Really great bitesie content. From what I remember from my cancer biology days (several years ago now!) is it also important to include several controls other than the loading control? i.e. A positive control that shows that the pull down of your protein of interest (in your example this would be protein A) has been successful to confirm an effective experimental setup etc; - so this would involve using an antibody against protein A too and if you have good band intensity in your western-blot then this would show that the antibody you're using/your experimental setup is sufficient. If you didn't use this positive control, any detected difference between results for other co-immunoprecipitant proteins might be a consequence of different levels of protein A. Am I right in thinking that an important negative control would be to also use an antibody that is of the same class (e.g. IgG if this was also used in the test experiments) but does not have specificity for the pull down protein target (Protein A in this case)? If we find that we don't detect the co-immunoprecipitant proteins of interest in the western-blot for this control, we can be more confident that the co-immunoprecipitated proteins in the test experiments have not bound to the FC region of the antibody and are therefore either directly (bound to protein A) or indirectly (bound to a protein which itself is bound to protein A) bound to protein A.

[@animatedbiologywitharpan]

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[@maymaung476]

Thanks a lot. your videos are really helpful for young researchers.

[@animatedbiologywitharpan]

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[@fabriciolima401]

thanks this is short, concise and logical, made it easy to understand

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[@paulchang8743]

how do you justify the pH of the buffer to make sure that protein A and B are not dissociated?

[@DiemNguyen-gg8gy]

Thank you so much for the clear explanation! You saved my course!!!!

[@animatedbiologywitharpan]

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[@hadeelkhalouf2104]

a very straightforward video ! thanks a lot !

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[@thomasprovoost7119]

Do you have this information from a source? If so, which one? Can you send me the link please

[@thomasprovoost7119]

You liked my reaction, but you don't answer?

[@lamialime1984]

Im a medical student,Thank you so much for this video❤️❤️❤️❤️it helps a lott!

[@animatedbiologywitharpan]

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Do checkout my other playlists

[@swatijagani9716]

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 ?

[@animatedbiologywitharpan]

Atleast you need to have one known protein which you would pull down and look for interactors via mass spec

[@fatcammal]

OH MY GOD THIS IS SO GOOD!!!!!!!!!!!!!!!!!!!! THANK YOU!!!!!!!!!!!!!

[@animatedbiologywitharpan]

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.

[@fatcammal]

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[@luciacarreno1268]

thanks a lot! from Argentina!!

[@animatedbiologywitharpan]

Glad to hear that my video helped..... share among friends as well

[@xchen8660]

Love your hand drawing!

[@animatedbiologywitharpan]

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[@traveladventure1818]

Very nice presentation

[@suryakantsonwanii]

How we select antibody for a protein, How we can sure that our antibody interact with our protein??

[@animatedbiologywitharpan]

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[@evanmcsharry1015]

Where is protein A on the blot? How is A not blotted?

[@jacobi_official8590]

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

[@aleksandrasokolova5492]

Thanks for explanation! It was really helpful

[@animatedbiologywitharpan]

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[@InquilineKea]

does this only work for covalent interactions?

[@animatedbiologywitharpan]

No no generally protein protein interactions are non covalent.

[@sksahidurrahaman7925]

Nicely explained explained. Could have been better if the importance of running input in the same gel was also given.

[@animatedbiologywitharpan]

I agree with your suggestion.

[@fabriciolima401]

actin is used just to make sure the proteins you are testing presence for were loaded correctly on the blot?

[@animatedbiologywitharpan]

Actin is a housekeeping protein so it’s not expected to change

[@netaburnum1359]

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.

[@Veedhi_Solanki]

very helpful video, the only video explains co-IP with western blot!

[@animatedbiologywitharpan]

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[@fahimakhan2621]

Explained very clearly thank you!

[@animatedbiologywitharpan]

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[@solimanalobaid6290]

Helpful 👌

[@animatedbiologywitharpan]

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[@caillouaudacieux]

Super useful, thank you !!

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[@jasonxlll5816]

Lourd la vidéo, ça m'a mis bien 👍

[@koy3080]

tell me are u from India? what is that accent??? i need to know

[@animatedbiologywitharpan]

Yes I am Indian, sorry if my accent has bothered you.

[@AZ-qx1xd]

thank you!

[@animatedbiologywitharpan]

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[@lukeshwarishriwas2896]

Sir it is in vitro technique????

[@animatedbiologywitharpan]

Could be used for in vitro or in vivo

[@emanelkafoury6891]

thank you

[@animatedbiologywitharpan]

Please share among your friends

[@jacknicholls4514]

So helpful, thanks!

[@animatedbiologywitharpan]

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[@marioalbertoleosramirez1569]

THANKS! NOW I CAN UNDERSTAND A PAPER THAT I HAVE TO READ xD

[@animatedbiologywitharpan]

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[@giuliablandino4526]

It's fantastic thanks!

[@animatedbiologywitharpan]

Most welcome! Please share with your friends to help my channel grow...😇

[@eyupyondem4818]

Thank you man, it will facilitate my presentation. You are the best :))

[@animatedbiologywitharpan]

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[@Segimaru]

Thank you for this video.

[@animatedbiologywitharpan]

Most welcome! Please share with your friends to help my channel grow...😇

[@arianahouman7914]

Thank you, sir :)

[@animatedbiologywitharpan]

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[@rajeshpalgp120]

good job brah...keep uploading

[@hopeaddict1322]

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[@animatedbiologywitharpan]

I am from TIFR

[@johnwilliams5064]

Thanks

[@aadam3657]

thank you brother!

[@firdausahmed581]

What is the purpose of using beta actin in this assay sir?

[@animatedbiologywitharpan]

It's a house keeping control......you can imagine it to be standard scale

[@usernamenishta-4411]

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.

[@animatedbiologywitharpan]

Nope ...you got it wrong..... this method can be used to detect both in vitro and in vivo protein protein interaction....

[@animatedbiologywitharpan]

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....

[@animatedbiologywitharpan]

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.....

[@usernamenishta-4411]

@@animatedbiologywitharpan ohhh okay. Yes that clears the confusion. Thankyouu 😊😊

[@usernamenishta-4411]

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[@michaelagunther4800]

great explanation

[@animatedbiologywitharpan]

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[@animatedbiologywitharpan]

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you save me!

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[@AlvinCwk]

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[@animatedbiologywitharpan]

It depends on the audiance

[@Asparagus-T]

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

[@animatedbiologywitharpan]

Email me at [e-mail]. we can interact further

[@faakehakhan7460]

what is this accent ?

[@animatedbiologywitharpan]

fakeha khan this is Bengali accent....sorry for that

[@tesconstamylo]

so by this way you describe yo identify the complex AB not their interaction as say !!!

[@biaohuanzhou4224]

good presetation, except some accent, but it's good. thanks.

[@secretsocietyserver]

Thanks!!

[@animatedbiologywitharpan]

Welcome!