Histone H3 Antibody (Tri-Methyl-Lys4) (OADC00277)

• Product Number: OADC00277
• Name: Histone H3 Antibody (Tri-Methyl-Lys4) (OADC00277)
• Conjugation: Unconjugated
• NCBI Gene Id: 8290; 8350; 8351; 8352; 8353; 8354; 8355; 8356; 8357; 8358; 8968
• Host: Rabbit
• Clonality: Polyclonal
• Concentration: 1 mg/mL
• Gene Full Name: histone cluster 3, H3
• Alias Symbols: H3t, H3.4, H3/g, H3FT, HIST3H3
• Product Format: Liquid. Whole antiserum from rabbit containing 0.05% azide.
• 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 4 (H3K4me3), using a KLH-conjugated synthetic peptide.
• Application Info: CHIP: 1 ul/CHIP
  ELISA: 1:100 - 1:500
  Dot Blotting: 1:20,000
  Western Blotting: 1:1,000
• 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-Lys4
• Preservative: 0.05% Sodium Azide
• Gene Symbol: HIST3H3
• Predicted Species Reactivity: Human
• Application: CHIP, DB, WB
• Image 1: Human Hela cells
  
  ChIP results obtained with the Aviva antibody directed against H3K4me3r. ChIP assays were performed using human HeLa cells, the Aviva antibody against H3K4me3 and optimized PCR primer pairs for qPCR. ChIP was performed with the "Auto Histone ChIP-seq" kit, using sheared chromatin from 1 million cells. A titration consisting of 1, 2, 5 and 10 ug of antibody per ChIP experiment was analyzed. IgG (2 ug/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the promoter of the active genes GAPDH and EIF4A2, used as positive controls, and for exon 2 of the inactive myoglobin (MB) gene and the Sat2 satellite repeat, used as negative controls. Figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that trimethylation of K4 at histone H3 is associated with the promoters of active genes.
• Image 2: HelaS3 cells
  
  ChIP-seq results obtained with the Aviva antibody directed against H3K4me3r. ChIP was performed on sheared chromatin from 1 million HeLaS3 cells using 1 ug of the Aviva antibody against H3K4me3 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 ELAND algorithm. Figure shows the peak distribution along the complete sequence and a 600 kb region of the X-chromosome and in two regions surrounding the GAPDH and EIF4A2 positive control genes, respectively. These results clearly show an enrichment of the H3K4 trimethylation at the promoters of active genes.
• Image 3: H3K4me3r
  
  Cross reactivity tests using the Aviva antibody directed against H3K4me3r. Figure 4A. To test the cross reactivity of the Aviva antibody against H3K4me3, a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H3K4. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:10,000. 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 a high specificity for the peptides containing the H3K4me3 modification.
• Image 4: H3K4me3
  
  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 H3K4me3. 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 1:5,300.
• Image 5: Human osteosarcoma (U2OS) cells
  
  Immunofluorescence using the Aviva antibody directed against H3K4me3r. Human osteosarcoma (U2OS) cells were stained with the Aviva antibody against H3K4me3 and with DAPI. Cells were fixed with 4% formaldehyde for 20' and blocked with PBS/TX-100 containing 5% normal goat serum. The cells were immunofluorescently labeled with the H3K4me3 antibody (top) diluted 1:200 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa568 or with DAPI (bottom), which specifically labels DNA.
• Image 6: Human Hela cells
  
  Western blot analysis using the Aviva antibody directed against H3K4me3r. Western blot was performed on whole cell (40 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 H3K4me3. 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.