H3K9me3 recombinant antibody and negative control
Diagenode is pleased to offer the first recombinant antibody against H3K9me3. It has has been selected using tailored phage-display libraries, and produced in E.coli eliminating animals from the antibody-production process. This recombinant antibody shows superior specificity and affinity as well as no lot-to-lot variation.
| Lot | 001 |
|---|---|
| Concentration | 0.25 µg/µl |
| Species reactivity | Human, mouse, drosophila, yeast |
| Type | Monoclonal, Recombinant |
| Purity | affinity purified |
| Host | Fab format with human framework |
| Precautions | This product is for research use only. Not for use in diagnostic or therapeutic procedures. |
| Applications | Suggested dilution/amount | References |
|---|---|---|
| ChIP/ChIP-seq * | 0.2-1.8 μg per ChIP | Fig 1, 2 |
| Immunofluorescence | 1:500 | Fig 3 |
* Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 0.5-5 μg per IP.
- 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)
Validation Data
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.
Material required
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.
Target Description
Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called “histone code”. Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases. Trimethylation of histone H3K9 is associated with imprinted regions, satellite repeats and ZNF repeat genes.
Applications
| IF Immunofluorescence: Diagenode offers huge selection of highly sensitive antibodies validated in IF. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9 HeLa cells transfected with a Cas9 expression vector (... Read more |
| ChIP-seq (ab) Read more |
| ChIP-qPCR (ab) Read more |
Documents
| Datasheet H3K9me3 C15500003 DATASHEET Datasheet description | Download |
| Antibodies you can trust POSTER Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar... | Download |
| Epigenetic Antibodies Brochure BROCHURE More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e... | Download |
Safety sheets
| H3K9me3 recombinant antibody and negative control SDS US en | Download |
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Publications
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H3K9 dimethylation safeguards cancer cells against activation of theinterferon pathway. |
SUV39 SET domains mediate crosstalk of heterochromatic histone marks. |
A conserved RNA degradation complex required for spreading and epigenetic inheritance of heterochromatin. |
Genomic Profiling by ALaP-Seq Reveals Transcriptional Regulation by PML Bodies through DNMT3A Exclusion. |
Abo1 is required for the H3K9me2 to H3K9me3 transition in heterochromatin. |
Native Chromatin Proteomics Reveals a Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance. |
Replication timing and epigenome remodelling are associated with the nature of chromosomal rearrangements in cancer. |
Automethylation-induced conformational switch in Clr4 (Suv39h) maintains epigenetic stability. |
Epigenetic inheritance mediated by coupling of RNAi and histone H3K9 methylation. |
Unique roles for histone H3K9me states in RNAi and heritable silencing of transcription |