TROPLIST: ChIP in embryos

[Hajime Ogino]

Hi Kurt,

I spent my whole summer to set up ChIP assay in Xenopus laevis embryos
(stages 22-24) using Upstate EZ ChIP kit.  The following points that I found
may be helpful for you.

(1) I fix embryos (vitelline membrane was removed) for 15 min at RT using 1%
formaldehyde/0.5xMBS.  This condition works well.  When the fixation time
was increased to 30 min, the chromatin became resistant to sonication.

(2) To remove yolk proteins that may cause high background, I firstly
sonicate tissues briefly without SDS and pellet the yolk by centrifugation.
SDS is then added to the supernatant to make the final conc. 1%, and this
chromatin sample is sonicated again for further shearing.  The first
sonication releases chromatin DNA from cells, but is not sufficient to make
their size short enough. The second sonication with SDS efficiently shear
the chromatin DNA (the size becomes 200 - 1000 bp).  After the second
sonication, I follow the protocol in the kit.

(3) Abcam anti-Otx2 antibody (ab21990) and anti-Myc tag antibody (9E10) work
well in my hands.

I remove vitelline membrane from embryos to increase penetration of the
fixative.  However, I do not like this laborious step.  Does anyone do ChIP
assay without removing the vitelline membrane?  I am happy if we could
communicate each other in the trop list to develop easier Xenopus ChIP
protocol.

Best,
Hajime Ogino
-----------------------------
Hajime Ogino, Ph.D.
Department of Biology
University of Virginia
Charlottesville
VA22904, U.S.A.
E-mail  ho8n@virginia.edu
Phone   +1-434-982-5605/5606
Fax     +1-434-982-5626





> I'm planning to perform some chromatin immunoprecipitations using
> trop embryos, but we have never ventured into this territory so I'd
> like to get any advice people may have to offer. I am planning to use
> the Upstate ChIP kit.
> 
> 1) Does anyone have any experience using this kit?
> 2) What protocol do people use to harvest the starting material prior
> to cross-linking? I'm planning to use st 22-28 embryos, but I'm
> guessing it would be similar to that used for other stages.
> 3) What cross-linking protocol do you use? Does it differ from
> standard protocols designed for culture cells?
> 4) What antibodies have you had success with?
> 
> Thanks,
> Kurt
> 
> 
> 
> 
> 
> Kurt Marek, Ph.D.
> 
> Division of Biological Sciences, Neurobiology Section
> University of California, San Diego
> 9500 Gilman Dr., Pacific Hall 3222B
> Mail Box 0357
> La Jolla, CA 92093
> Ph: 858-534-2456
> kmarek@ucsd.edu
> 
>