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Immunoprecipitation and immunobloting with different antibodies


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6 replies to this topic

#1 feifei83

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Posted 16 March 2011 - 01:36 PM

I used one rabbit polyclonal antibody and one mouse monoclonal antibody to IP the same protein from cell lysate. Then use these two antibodies to detect the IP product. Both of the two antibodies worked well for immunobloting of the total cell lysate--a clear band at the position of the protein's MW. However, only IP/IB with the same antibody worked. That means, I could use Rabbit Ab to IB the IP product from Rabbit Ab but not from Mouse Ab. That was the same with mouse Ab.
The Rabbit Ab is said to be raised against the AA 1-70 of this protein, and the mouse ab is against the FL of the protein.
I thought IP with different antibodies should precipitate the same FL protein. Why IP with Rabbit and IB with Mouse did not work? Also the vice versa.
Thanks.

#2 bob1

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Posted 20 March 2011 - 04:34 PM

It could be that one (or both) of the antibodies is not very efficient at immunoprecipitating you protein of interest, so you can't detect it on a blot. Are you sure that the band(s) you are seeing on your IP/IB with the same antibody are not heavy chain or light chain IgG?

#3 feifei83

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Posted 22 March 2011 - 07:46 AM

It could be that one (or both) of the antibodies is not very efficient at immunoprecipitating you protein of interest, so you can't detect it on a blot. Are you sure that the band(s) you are seeing on your IP/IB with the same antibody are not heavy chain or light chain IgG?

Thanks.
The band is at about a little more than 75kD. Also the WB of this protein is at this position (I run IP and total lysate on the same gel). Could it be IgG heavy + light chain (75+25kD)?

#4 bob1

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Posted 27 March 2011 - 03:57 PM

If you are using a denaturing blot and boiling your samples in denaturing loading buffer, it is very unlikely that the heavy and light chains are still joined. If your lysate is running at the correct size, it is likely that the antibodies are not very good. Monocolonal antibodies are often not very good at immunoprecipitating.

Are you sure that your antibodies will detect native protein like you would have in an IP?

#5 chouchou

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Posted 31 March 2011 - 09:56 AM

I kinda have a similar problem. I used a monoclonal AB against a tag on my protein for IP, and then if I use the same mono-AB for IB it worked fine, but if a different poly-AB againt part of my protein was used for IB, I could not see my protein. How does this happen?

#6 bob1

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Posted 31 March 2011 - 04:01 PM

Does the polyclonal recognise denatured protein? You will be better off IPing with the polyclonal and detecting with the monoclonal.

#7 chouchou

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Posted 31 March 2011 - 05:52 PM

Does the polyclonal recognise denatured protein? You will be better off IPing with the polyclonal and detecting with the monoclonal.


I'm not sure. But it might be the problem. I'll try IPing with polyclonal to see. Thanks.




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