Histone H2A Antibody (Acetyl-Lys5/7/11) (OADC00064)

• Product Number: OADC00064
• Name: Histone H2A Antibody (Acetyl-Lys5/7/11) (OADC00064)
• Conjugation: Unconjugated
• NCBI Gene Id: 3012; 317772; 8335; 8338
• Host: Rabbit
• Clonality: Polyclonal
• Concentration: 0.7 mg/mL
• Gene Full Name: histone cluster 1, H2ae
• Alias Symbols: H2A.1, H2A.2, H2A/a, H2AC4, H2AFA, HIST1H2AE
• 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 H2A family. Transcripts from this gene lack polyA tails; instead, they contain a palindromic termination element. This gene is found in the large 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 histone H2A.Z acetylated at lysines 5, 7 and 11, using a KLH-conjugated synthetic peptide.
• Application Info: CHIP: 1 ug/CHIP
  ELISA: 1:200
  Dot Blotting: 1:20,000
  IF: 1:500
• Uniprot ID: P0C0S5
• Protein Name: histone H2A type 1-B/E
• Protein Accession #: NP_002097.1
• Purification: Affinity purified
• Nucleotide Accession #: NM_002106.3
• Target Post-Translational Modification: Acetyl-Lys5/7/11
• Preservative: 0.05% Sodium Azide and 0.05% ProClin 300.
• Gene Symbol: HIST1H2AE
• Predicted Species Reactivity: Human
• Application: CHIP, DB, IF
• Image 1: Human Hela cells
  
  ChIP results obtained with the Aviva antibody directed against H2A.Zacr. ChIP assays were performed using HeLa cells, the Aviva antibody against H2A.Zac and optimized primer pairs for qPCR. ChIP was performed on sheared chromatin from 1 million HeLaS3 cells using the "Auto Histone ChIP-seq" kit on the SX-8G IP-Star automated system. A titration of the antibody consisting of 1, 2, 5 and 10 ug per ChIP experiment was analysed. IgG (2 ug/ IP) was used as negative IP control. QPCR was performed using primers specific for the promoters of the ACTB and EIF4A2 genes, used as positive control targets and for the coding region of the MYT1 gene, used as a negative control target. Figure shows the recovery (the relative amount of immunoprecipitated DNA compared to input DNA). These results confirm the observation that acetylation of H2A.Z is present at active promoters.
• Image 2: HelaS3 cells
  
  ChIP-seq results obtained with the Aviva antibody directed against H2A.Zacr. ChIP was performed with 1 ug of the Aviva antibody against H2A.Zac on sheared chromatin from 1 million HeLaS3 cells. IgG (2 ug/IP) was used as a negative IP control. The IP'd DNA was analysed by QPCR as described above. The IP'd DNA was subsequently analysed with 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 100 kb regions surrounding the EIF4A2, ACTB and GAPDH genes. These results clearly show an enrichment of the H2A.Z acetylation at the promoters of active genes.
• Image 3: H2A.Zacr
  
  Cross reactivity test using the Aviva antibody directed against H2A.Zacr. A Dot Blot analysis was performed to test the cross reactivity of the Aviva antibody against H2A.Zac with peptides containing other histone acetylations and the unmodified H2A.Z sequence. 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 4 shows a high specificity of the antibody for the modification of interest.
• Image 4: H2A.Zac
  
  Determination of the antibody titer. To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Aviva antibody directed against H2A.Zac, crude serum and flow through in antigen coated wells. The antigen used was a peptide containing the histone modifications of interest. By plotting the absorbance against the antibody dilution, the titer of the purified antibody was estimated to be 1:8,800.
• Image 5: Hela cells
  
  Immunofluorescence using the Aviva antibody directed against H2A.Zacr. HeLa cells were stained with the Aviva antibody against H2A.Zac 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 labelled with the H2A.Zac antibody (left) 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 on the right.