Comments on: Co-IP and ChIP in Plants http://www.cshblogs.org/cshprotocols/2008/09/09/co-ip-and-chip-in-plants/ Tue, 6 Jan 2009 01:49:36 +0000 http://wordpress.org/?v=2.6.3 By: David Crotty http://www.cshblogs.org/cshprotocols/2008/09/09/co-ip-and-chip-in-plants/#comment-16741 David Crotty Sun, 07 Dec 2008 17:28:22 +0000 http://www.cshblogs.org/cshprotocols/2008/09/09/co-ip-and-chip-in-plants/#comment-16741 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! 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!

]]> By: David Crotty http://www.cshblogs.org/cshprotocols/2008/09/09/co-ip-and-chip-in-plants/#comment-16731 David Crotty Wed, 03 Dec 2008 17:25:48 +0000 http://www.cshblogs.org/cshprotocols/2008/09/09/co-ip-and-chip-in-plants/#comment-16731 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). 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).

]]> By: Andrea Hricova http://www.cshblogs.org/cshprotocols/2008/09/09/co-ip-and-chip-in-plants/#comment-16729 Andrea Hricova Wed, 03 Dec 2008 12:14:49 +0000 http://www.cshblogs.org/cshprotocols/2008/09/09/co-ip-and-chip-in-plants/#comment-16729 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 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

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