Cross-linking (X-ChIP) Duration of cross-linking step: 0 second(s) Specification of the cross-linking agent: 3% Formaldehyde
I specifically induce silencing of the ura4+ gene. I see enrichment for this gene in ChIPs performed with antibodies against H3K9me2, Ago1, Swi6 and, as I show here, Chp1. All these proteins are recruited to transcriptional silent loci as for example centromeric DNA repeats. The Chp1 antibody precipitates chromatin very well (see figure )-it gives me the strongest signal among all the antibodies I used so far.
I perform multiplex PCR with primers amplifying ura4+ and an un-silenced gene. In addition, act1+ was amplified in a separate PCR reaction. As you can see, Chp1 only binds to ura4+ (and only if silencing is induced).
Other product details
I used 5ul antibody per 500ul of chromatin/cell lysate according to standard S. pombe ChIP protocols. (50ml cells at OD600=1.3 were crosslinked, washed and frozen in liquid N2. Crude lysate was prepared with 360 microliter lysis buffer. About one third of the crude lysate was used for the Chp1 ChIP.) Protein-A Sepharose from Amersham was used to recover the ChIPs.
Dr. Marc B?
Submitted Feb 09 2006
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