Histone H4 Antibody (Tri-Methyl-Lys20) (OADC00262)

Data Sheet
 
Product Number OADC00262
Product Page www.avivasysbio.com/histone-h4-antibody-tri-methyl-lys20-oadc00262.html
Name Histone H4 Antibody (Tri-Methyl-Lys20) (OADC00262)
Conjugation Unconjugated
NCBI Gene Id 121504; 554313; 8294; 8359; 8360; 8361; 8362; 8363; 8364; 8365; 8366; 8367; 8368; 8370
Host Rabbit
Clonality Polyclonal
Concentration 0.94 mg/mL
Gene Full Name histone cluster 4, H4
Alias Symbols H4/p, H4C1, H4C2, H4C3, H4C4, H4C5, H4C6, H4C8, H4C9, H4C11, H4C12, H4C13, H4C14, H4C15, HIST4H4
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 H4 family. Transcripts from this gene lack polyA tails; instead, they contain a palindromic termination element.
Reconstitution and Storage Store at -20C. Avoid repeated freeze/thaw cycles.
Datasheets/Manuals Printable datasheet for anti-HIST4H4 (OADC00262) antibody
Specificity Polyclonal antibody raised in rabbit against the region of histone H4 containing the trimethylated lysine 20 (H4K20me3), using a KLH-conjugated synthetic peptide. 
Application Info CHIP: 0.5-1 ug per IP
ELISA: 1:500
Dot Blotting:/Peptide array 1:20,000/1:10,000
Western Blotting: 1:1,000
Immunofluorescence: 1:500
Protein Name histone H4
Purification Affinity purified
Target Post-Translational Modification Tri-Methyl-Lys20
Preservative 0.05% Sodium Azide and 0.05% ProClin 300.
Gene Symbol HIST4H4
Predicted Species Reactivity Human
Application CHIP, DB, WB, IF
Predicted Homology Based on Immunogen Sequence Yeast
Image 1
Human K562 cells
ChIP results obtained with the Aviva antibody directed against H4K20me3r. ChIP assays were performed using human K562 cells, the Aviva antibody against H4K20me3 and optimized PCR primer sets for qPCR. ChIP was performed with the "iDeal ChIP- seq" kit on sheared chromatin from 100,000 cells. A titration of the antibody consisting of 0.2, 0.5, 1 and 2 ug per ChIP experiment was analysed. IgG (1 ug/IP) was used as negative IP control. QPCR was performed with primers for promoters of the active genes EIF4A2 and GAPDH, used as negative controls, and for ZNF510 and the Sat2 satellite repeat region used as positive controls. 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 H4K20me3r. ChIP was performed with 0.5 ug of the Aviva antibody against H4K20me3 on sheared chromatin from 100,000 K562 cells. The IP'd DNA was analysed by QPCR as described above (figure 2A). 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 signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figures show the enrichment in the telomeric region of chromosome 12, also containing several ZNF repeat genes, and at ZNF510 on chromosome 9, respectively. The position of the amplicon used for ChIP-qPCR is indicated by an arrow.
Image 3
H4K20me3r
Cross reactivity tests using the Aviva antibody directed against H4K20me3r. Figure 4A To test the cross reactivity of the Aviva antibody against H4K20me3, a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4K20. 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 1:10,000. 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
H4K20me3
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 H4K20me3 in antigen coated wells. 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 1:21,700.
Image 5
Hela cells
Immunofluorescence using the Aviva antibody directed against H4K20me3r. HeLa cells were stained with the Aviva antibody against H4K20me3 and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K20me3 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.
Image 6
Human Hela cells
Western blot analysis using the Aviva antibody directed against H4K20me3r. 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 H4K20me3. 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.
 

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