ChIP assays were performed using human HeLa cells. A titration of the antibody consisting of 0.5, 1, 2 and 5 ug per ChIP experiment was analysed. IgG (1 ug/IP) was used as negative IP control. QPCR was performed with primers for a region approximately 1 kb upstream of the ACTB and GAPDH promoters, used as positive controls, and for the coding region of the inactive MYOD1 gene and the Sat2 satellite repeat, used as negative controls. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
Western Blot (Cell lysate)
Western Blot (Cell lysate) analysis of (1) 25 ug whole cell extracts of HeLa cells, (2) 15 ug histone extracts of HeLa cells, (3) 1 ug of recombinant histone H2A, (4) 1 ug of recombinant histone H2B, (5) 1 ug of recombinant histone H3, and (6) 1 ug of recombinant histone H4.
Enzyme-linked Immunoabsorbent Assay
ELISA is a quantitative method used to determine the titer of the antibody using a serial dilution of antibody against Histone H3 (K14ac) 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 1:73200.
Dot Blot
Cross reactivity tests using the Histone H3 (K14ac) antibody. Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H3K14. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:10000. The figure shows a high specificity of the antibody for the modification of interest.
Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. This structure consists of approximately 146 bp of DNA wrapped around a nucleosome, an 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 member of the histone H3 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. [provided by RefSeq