5 hour(s) and 0 minute(s) · Diluent: 1% BSA in PBS
The real application is sequential ChIP or re-ChIP. First IP was perfomed with anti H3 and later on Str Beads were used to do a new IP, before QT-PCR analysis.
H3 oxidation in major satellite region was
determined by sequential ChIP (re-ChIP) in control
and SNA1 KO MEFs. The lysate was incubated with
biotin-hydrazide (an activated biotin that reacts
with oxidized H3) before re-ChIP. Extracts were
sequentially immunoprecipitated with anti-H3 and
streptavidin beads. DNA binding was quantified
by qPCR. Data were normalized to the total
amount of H3 immunoprecipitated and to the input
and expressed as fold enrichment over the data
obtained when an irrelevant IgG was used.
To detect H3 oxidation,
ChIP assays were performed as described previously (Herranz et al., 2012).
Briefly, cell extracts were incubated with activated biotin, crosslinked, and
immunoprecipitated with anti-histone H3 immunocomplexes were reextracted
with SDS lysis buffer and reimmunoprecipitated with streptavidinmagnetic
beads for 30 min at 4C. Samples were then treated with elution
buffer and incubated at 65C to reverse formaldehyde crosslinking. Results
were quantified by taking into account the total amount of H3 immunoprecipitated
in each condition.