Co-IP and ChIP in Plants
Tuesday, September 9, 2008 at 10:40 am CDT by David Crotty permalink
John Mundy’s laboratory at the University of Copenhagen has written up protocols for Coimmunoprecipitation (co-IP) of Nuclear Proteins and Chromatin Immunoprecipitation (ChIP) from Arabidopsis in the September issue of CSH Protocols. co-IP is useful for identifying and isolating protein-protein interactions and protein complexes. ChIP allows the analysis of protein-DNA interactions, and is a technique currently seeing widespread use as the field of transcriptional regulation continues to make great advances. One advantage to this set of techniques is that the nuclear lysis buffers standard in most protocols for plant nuclear protein extraction are incompatible with co-IP. Here, nuclear protein extraction is accomplished via sonication in co-IP buffer and treatment with Benzonase, which results in material that can be used with co-IP.
Posted in Cell Biology, General, Laboratory Organisms, Molecular Biology, Plant Biology, Proteins and Proteomics |
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Wednesday, December 3, 2008 at 6:14 am CST
Hi, I would appreciate some comment on the protein concentration and lysate sonication. I work with Arabidopsis nuclear protein. After sonication I measure the concentration of (nuclear) proteins in lysate and it is really very low to perform further steps and end up with detection. I know that if sonication is not set up well, that can be harsh and distrupt also protein interactions one is interested in. What do you recommend as the optimal sonication, or what buffer are best for this step? Is the high amount of plant tisue really so critical? Some papers decribe to start with 25 up to 100(!) g of Arabidopsis tissue. I have used last time 5 g of plant tissue and end up with 0.2-0.4 mikrograms of protein per mikroliter.
Thanks a lot for helpful comments and advices.
Sincerely Andrea Hricova
Wednesday, December 3, 2008 at 11:25 am CST
Hi Andrea,
I’ve put up your question as a comment on the protocol in question, as it may be of interest to others who are using this technique. I’ve also forwarded it along to the authors, and I’ll let you know when I receive an answer (and will post it here).
Sunday, December 7, 2008 at 11:28 am CST
Andrea, here’s an answer from John Mundy, who wrote the protocol. Hope this helps:
-We have worked with nuclear proteins present at low levels (Andreasson et al. 2005 EMBOJ 24, 2579 and Qiu et al 2008 EMBOJ 27, 2214). We have satisfactory experience with 70-100 g of tissue which gives us a yield of approximately 1 ug protein per ul in a total volume of ~300 ul. The amount of tissue/ protein required for an experiment is of course dependent on the level of the protein of interest.
-Changing sonicator from the one we used in our protocol requires optimization of the sonication step. The sonication should be determined empirically by e.g. running SDS PAGE on crude extracts and blotting against your protein of interest after different sonication times. When sonicating, apply short pulses, and let the sample cool in between sonications. It is very important to keep temperature low, so perform sonication in ice bath.
-Sonication buffers should include HEPES or Tris pH 7.4-8, EDTA, Triton-X, salt (NaCl), SDS and protease inhibitors. Exact concentrations vary from protocol to protocol. SDS improves sonication efficiency, but high levels interfere with the antibodies (http://www.epigenome-noe.net/researchtools/protocol.php?protid=10).
We have recommended a buffer 100 mM Tris.Cl, pH 7.4, 75 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 0.05% SDS, 10% glycerol as it is compatible with both sonication and immunoprecipitation.
-As in all empirical endeavours, good luck!