Histone H3 Antibody (Tri-Methyl-Lys9) (OADC00255)

• Product Number: OADC00255
• Name: Histone H3 Antibody (Tri-Methyl-Lys9) (OADC00255)
• Conjugation: Unconjugated
• NCBI Gene Id: 8290; 8350; 8351; 8352; 8353; 8354; 8355; 8356; 8357; 8358; 8968
• Host: Rabbit
• Clonality: Polyclonal
• Concentration: 2.7 mg/mL
• Gene Full Name: histone cluster 3, H3
• Alias Symbols: H3t, H3.4, H3/g, H3FT, HIST3H3
• Product Format: Liquid. Provided in PBS containing 0.05% azide and 0.05% ProClin 300.
• Description of Target: Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. Nucleosomes consist of approximately 146 bp of DNA wrapped around a histone octamer composed of pairs of each of the four core histones (H2A, H2B, H3, and H4). The chromatin fiber is further compacted through the interaction of a linker histone, H1, with the DNA between the nucleosomes to form higher order chromatin structures. This gene is intronless and encodes a replication-dependent histone that is a member of the histone H3 family. Transcripts from this gene lack polyA tails; instead, they contain a palindromic termination element. This gene is located separately from the other H3 genes that are in the histone gene cluster on chromosome 6p22-p21.3.
• Reconstitution and Storage: Store at -20C. Avoid repeated freeze/thaw cycles.
• Specificity: Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylated lysine 9 (H3K9me3), using a KLH-conjugated synthetic peptide.
• Application Info: CHIP: 1 ug per IP
  ELISA: 1:5,000
  Dot Blotting:/Peptide array 1:20,000/1:2,000
  Western Blotting: 1:1,000
  IF: 1:500
• Uniprot ID: Q16695
• Protein Name: histone H3.1t
• Protein Accession #: NP_003484.1
• Purification: Affinity purified
• Nucleotide Accession #: NM_003493.2
• Target Post-Translational Modification: Tri-Methyl-Lys9
• Preservative: 0.05% Sodium Azide and 0.05% ProClin 300.
• Gene Symbol: HIST3H3
• Predicted Species Reactivity: Human
• Application: CHIP, DB, WB, IF
• Predicted Homology Based on Immunogen Sequence: Yeast
• Image 1: Human Hela cells
  
  ChIP results obtained with the Aviva antibody directed against H3K9me3r. Figure 1.A ChIP assays were performed using human HeLa cells, the Aviva antibody against H3K9me3 and optimized PCR primer pairs for qPCR. ChIP was performed using sheared chromatin from 1,000,000 cells. A titration consisting of 0.2, 0.5, 1, 2 and 5 ug of antibody per ChIP experiment was analyzed. IgG (1 ug/IP) was used as a negative IP control. Figure 1.B ChIP assays were performed using human K562 cells, the Aviva antibody against H3K9me3 and optimized PCR primer pairs for qPCR. ChIP was performed with the "iDeal ChIP-seq" kit, using sheared chromatin from 100,000 cells. A titration consisting of 0.2, 0.5, 1 and 2 ug of antibody per ChIP experiment was analyzed. IgG (1 ug/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the promoter of the active genes GAPDH, c-fos and EIF4A2, used as negative controls, and for ZNF510 and the Sat2 satellite repeat, used as positive controls. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
• Image 2: K562 cells
  
  ChIP-seq results obtained with the Aviva antibody directed against H3K9me3r. ChIP was performed on sheared chromatin from 100,000 K562 cells using 1 ug of the Aviva antibody against H3K9me3 with the iDeal ChIP-seq kit as described above. The IP'd DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 36 bp tags were aligned to the human genome using the BWA algorithm. Figure 2A shows the H3K9me3 signal distribution along the long arm of human chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2B shows the signal distribution in a 200 kb region from chromosome 12 surrounding the ZNF12 gene.
• Image 3: H3K9me3r
  
  Cross reactivity tests using the Aviva antibody directed against H3K9me3r. Figure 4A. To test the cross reactivity of the Aviva antibody against H3K9me3, a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H3K9. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4A shows a high specificity of the antibody for the modification of interest. Figure 4B. The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:2,000. Figure 4B shows the specificity factor, calculated as the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark.
• Image 4: H3K9me3
  
  Determination of the antibody titer. To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Aviva antibody against H3K9me3. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:115,000.
• Image 5: Hela cells
  
  Immunofluorescence using the Aviva antibody directed against H3K9me3r. HeLa cells were stained with the Aviva antibody against H3K9me3 and with DAPI. Cells were fixed with 4% formaldehyde for 10' and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H3K9me3 antibody (top) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown at the bottom.
• Image 6: Human Hela cells
  
  Western blot analysis using the Aviva antibody directed against H3K9me3r. Western blot was performed on whole cell (25 ug, lane 1) and histone extracts (15 ug, lane 2) from HeLa cells, and on 1 ug of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Aviva antibody against H3K9me3. The antibody was diluted 1:1,000 in TBS-Tween containing 5% skim milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left.