10 replies to this topic

[A0110]

Hi all,
Have anyone tried immunoprecipitating two proteins simultaneously (in the same step) using two different primary antibodies? Does it work well or is it better to do each protein independently?

Thanks

[bob1]

How would you tell that both ppted and that it wasn't the result of a co-IP?  Do them separately.

[A0110]

Thanks for the reply.
If I will be analyzing it by western blot using specific Ab will that be fine?
What do you mean by co-IP?

If I will do them separate then I guess I would be reusing the supernatant after immunoprecipitation and re-precipitating the other protein of interest?! Would sepharose bind with my proteins and thus affect the outcome if IP was performed on the superntant?

[lab rat]

Co-ip means co-immunoprecipitation. Two proteins that associate can be pulled down together using an antibody specific for one of them.

I agree with Bob. Use two separate columns to pull down your two proteins. I think you would find that your yield of one or both proteins will be diminished if you try to pull them both down with the same column, even if you do one after the other.

You can re-use the flow-through from the first column to bind your second protein on the second column, as long as it contains only binding or washing buffer and not an elution buffer.

[A0110]

Thanks! I really appreciate your suggestions.

[ethan241]

Hello there!

I was wondering if it was possible to do an immunoprecipitation using one antibody, then doing a second with a phosphospecific antobody?  Wouldn't that pulldown only proteins that were associated with phosphospecific versions of your original protein of interest?

Thanks for your help!

[laurequillo]

ethan241, on 03 August 2010 - 10:47 AM, said:

Hello there!

I was wondering if it was possible to do an immunoprecipitation using one antibody, then doing a second with a phosphospecific antobody?  Wouldn't that pulldown only proteins that were associated with phosphospecific versions of your original protein of interest?

Thanks for your help!

Yeah sure, you cando that to check whats the pool of phosphorylated proteins you pool down with your first ab. For example you do the first Ip with flag,then elute with flag peptide, and then do the second ip with the phosphospecific one

[ethan241]

Hmmm, now that you mention that I'm not sure it would pull down what I was hoping for, I wanted to find proteins that would attach to a phosphorylated protein, not ones that were p'd themselves.  But I think if I do a pull down for SH2 regions in the second, that would give me ones that would be TYR phosphorylated. . . maybe. . . I'll have to see if there are ones for SER and THR too. . .

laurequillo, on 03 August 2010 - 12:15 PM, said:

ethan241, on 03 August 2010 - 10:47 AM, said:

Hello there!

I was wondering if it was possible to do an immunoprecipitation using one antibody, then doing a second with a phosphospecific antobody?  Wouldn't that pulldown only proteins that were associated with phosphospecific versions of your original protein of interest?

Thanks for your help!

Yeah sure, you cando that to check whats the pool of phosphorylated proteins you pool down with your first ab. For example you do the first Ip with flag,then elute with flag peptide, and then do the second ip with the phosphospecific one

[YeeNU]

I am trying to do a similar experiment but hoping to show that my 3 proteins of interest bind in a complex. But I am not sure how to elute the proteins from the beads after the first immunoprecipitation. I do not want to use reagents, such as SDA, that would disrupt my complexes. Any suggestions on how to troubleshoot this? Or suggestions on how to prove 3 proteins bind together in a complex.

Thank you.

[mdfenko]

you can often release a protein from antibody by reducing pH to below 3 (2.5 is often used). sometimes you may have to go to high pH (10-11).

you will have to determine if either of those conditions will disrupt your protein complex.

to prove the proteins form a complex you can determine the size of the complex by gel filtration.

[Arun Kumaran]

You can use 0.1 M NaCl (pH 11.3) to separate protein complex from antibody. After eluted, you have to neutralize it.

Edited by Arun Kumaran, 01 June 2012 - 10:08 AM.