H4-16 Rabbit Polyclonal Antibody

CAT#: TA347213

Rabbit Polyclonal H4K5,8,12,16ac Antibody

Product Images

Determination of the antibody titer To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody against H4K5, 8, 12, 16ac in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:21, 200.

• 

  INIH3T3 cells were stained with the ab against H4K5, 8, 12, 16ac 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 H4K5, 8, 12, 16ac 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.

• 

  A Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4. 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.

• 

  ChIP assays using HeLa cells (sheared chromatin from 1 million cells). Titration of 1, 2, 5 and 10ug antibody per ChIP was analysed. IgG (2 ug/IP) was used as negative control. qPCR primers were for promoter of active gene EIF4A2 and for a region 1 kb upstream of GAPDH as positive controls, and for inactive MYOD1 and the Sat2 satellite repeat region used as negative controls. Image shows the recovery, expressed as a % of input (the relative amount of IP'd DNA compared to input DNA after qPCR).

• 

  ChIP was performed with 2 ug of the ab on sheared chromatin from 1 million HeLa cells. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Image shows the signal distribution along the complete length of chromosome 5 and a zoomin to a 600 kb region (B). C and D show the enrichment in two genomic regions on chromosome 3 and 12, respectively, containing EIF4A2 and GAPDH positive controls. The position of the amplicon used for validating the qPCR results is shown with arrow.

Show all 5 images

Specifications

Product Data

• Applications: Dot, ELISA, IF
• Recommended Dilution: ChIP/ChIP-seq (2ug/IP); ELISA (1:1,000); Dot blotting (1:20,000); Immunofluorescence (1:500)
• Reactivities: Human, Mouse, Broad
• Host: Rabbit
• Isotype: IgG
• Clonality: Polyclonal
• Immunogen: The immunogen for anti-H4K5,8,12,16ac antibody: the region of histone H4 containing the acetylated lysines 5, 8, 12 and 16 (H4K5,8,12,16ac), using a KLH-conjugated synthetic peptide.
• Concentration: lot specific
• Purification: Affinity purified polyclonal antibody in PBS containing 0.05% azide and 0.05% ProClin 300.
• Conjugation: Unconjugated
• Storage: Store at -20°C as received.
• Stability: Stable for 12 months from date of receipt.
• Gene Name: histone cluster 4, H4
• Database Link:
  NP_778224
  Entrez Gene 320332 MouseEntrez Gene 121504 Human
  UniProt ID P62805
• Background: 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. Acetylation of histone H4 is associated with active genes.
• Synonyms: H4; p

Reference Data

• Protein Pathways: Systemic lupus erythematosus