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coIP followed by a Western Blot

coIP Western Blot

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4 replies to this topic

#1 Mad Researcher

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Posted 24 January 2018 - 09:24 AM

Hi,

 

I want to do a co-IP followed by a Western Blot, however, I am confused.

 

I use antibody A for doing a coIP. And I want to check the interaction of antibody A with Antibody B.

So, after the coIP steps, I am doing a Western with Antibody B. Is it necessary to do another Western with Antibody A, too?

Or will I see an interaction once I do a Western with Antibody B only? Also, is it necessary to do a loading control antibody too?

 

Thanks


Cheers,

Mad Researcher

#2 bob1

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Posted 24 January 2018 - 10:06 AM

Detecting with both antibodies is appropriate, as you want to demonstrate first that your basic IP (that of protein A) is working, then that the co-IP has happened.

 

It is unlikely that a loading control would work - your IP steps should remove most of the proteins that you would typically use for IP. Most people would state how much protein was loaded into the IP and the amount (ug) of antibody used at each step. You should however run a western blot for your proteins A and B to demonstrate that they are present in the input samples. For these blots you would use a loading control.



#3 Mad Researcher

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Posted 24 January 2018 - 11:32 AM

Detecting with both antibodies is appropriate, as you want to demonstrate first that your basic IP (that of protein A) is working, then that the co-IP has happened.

 

It is unlikely that a loading control would work - your IP steps should remove most of the proteins that you would typically use for IP. Most people would state how much protein was loaded into the IP and the amount (ug) of antibody used at each step. You should however run a western blot for your proteins A and B to demonstrate that they are present in the input samples. For these blots you would use a loading control.

So, once I have done blotting for antibody B, I should strip it off and probe for antibody A?

 

If I don't do it, will I still see protein interaction like in the attached figure?

Attached Thumbnails

  • 1.png

Edited by Mad Researcher, 24 January 2018 - 11:32 AM.

Cheers,

Mad Researcher

#4 bob1

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Posted 24 January 2018 - 01:45 PM

That's one way to do it. Personally I prefer to use a separate membrane for the different proteins, or try and have different species primary antibodies so that they can be detected on the same membrane, if protein sizes differ. Ideally you will be using different antibodies for the IP and detection steps. 



#5 Mad Researcher

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Posted 24 January 2018 - 02:44 PM

That's one way to do it. Personally I prefer to use a separate membrane for the different proteins, or try and have different species primary antibodies so that they can be detected on the same membrane, if protein sizes differ. Ideally you will be using different antibodies for the IP and detection steps. 

I don't have more protein to use another membrane.

So, I think I need to strip the membrane and repeat. Can you suggest any good stripping buffer?

 

Also, If I don't do it, will I still see protein interaction?


Cheers,

Mad Researcher





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