Figure 1. ChIP results obtained with the Diagenode recombinant antibody directed against H3K9me3
ChIP assays were performed using human HeLa cells, the Diagenode recombinant antibody against H3K9me3 and optimized PCR primer sets for qPCR. ChIP was performed on sheared chromatin from 100,000 and 5,000 cells with the “True MicroChIP kit (cat. No. C01010130). See page 4: Protocol for binding the recombinant H3K9me3 antibody to streptavidin- coated beads (Hattori T. et al., 2013). Different amounts of the antibody were analysed. A negative control recombinant antibody 1 or 5 μg/IP) was used as negative IP control. QPCR was performed with primers for the heterochromatin marker Sat2 and for the ZNF510 gene, used as positive controls, and for the promoters of the active EIF4A2 and GAPDH genes, used as negative controls. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
Figure 2. ChIP-seq results obtained with the Diagenode recombinant antibody directed against H3K9me3
ChIP was performed with 1.3 μg of the Diagenode antibody against H3K9me3 on sheared chromatin from 4 million K562 cells. The IP’d DNA was analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The sequenced reads were aligned to human genome version 19 using the ELAND algorithm. Figure 2A shows the signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2B shows the enrichment at ZNF510 and Figure 2 C and D show the enrichment at the MEG3 and KCNQ1 imprinted genes.
Figure 3. Immunofluorescence using the Diagenode recombinant antibody directed against H3K9me3
NIH 3T3 cells were stained with the Diagenode antibody against H3K9me3, left or with the negative control recombinant antibody, right. The bottom panel shows counterstaining of the cells with DAPI. (Hattori T. et al., 2013).
Protocol for binding the recombinant H3K9me3 antibody to streptavidin-coated beads (Hattori T. et al., 2013). The recombinant H3K9me3 antibody (Cat. No. C15500003) has been validated in ChIP with the True MicroChIP kit (Cat. No. C01010130). However, since this antibody is a biotinylated Fab fragment, the protocol was slightly adapted. The protein A/G coated magnetic beads included in the True MicroChIP kit were replaced by streptavidin-coated beads to capture the recombinant antibody. The protocol below is intended for binding of the antibody to streptavidin beads for one ChIP experiment. Scale up accordingly for larger numbers of ChIP experiments.
- Dynabeads M280 Streptavidin (Invitrogen)
Alternatively Streptavidin MagneSphere paramagnetic beads (Promega) can be used
- TBS containing 0.5% BSA (called TBS/BSA in the protocol)
- Biotin. Prepare a solution of 5 μM biotin in TBS containing 0.5 % BSA
- Diamag 1.5 magnetic rack (Cat No. kch-816-015)
NOTE: Please proceed with STEP 1 - Cell collection and DNA-protein crosslinking as well as STEP 2 - Cell lysis and chromatin shearing, as explained in the True MicroChIP kit protocol. In STEP 3 – Magnetic Immunoprecipitation and washes, proceed up to point 22 for Detailed protocol or point 13 for Short protocol (ie proceed up to chromatin dilution after the shearing and use this diluted chromatin at the end of the recombinant antibody binding protocol below).The protocol below is optimized for working with 100 000 cells. When using less cells, you should decrease the amount of antibody and beads to use.