The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Use 5 - 15 μl/ChIP for chromatin prepared from 1.5 million cells.
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 fibre is further compacted through the interaction of a linker histone, H1, with the DNA between the nucleosomes to form higher order chromatin structures.
Covalent modifications of the canonical core histones, including acetylation, phosphorylation, methylation, and monoubiquitination, are used to mark nucleosomes to create chromatin domains with a range of functions. The information encoded by the modifications can contribute to the formation and/or maintenance of transcriptionally active and inactive chromatin in response to various signalling pathways.
Phosphorylation, particularly that of histones H1 and H3, has long been implicated in chromosome condensation during mitosis. However, converging evidence suggests that H3 phosphorylation (specifically serine 10) is also directly correlated with the induction of immediate-early genes such as c-jun, c-fos and c-myc. The potential importance of the serine 10 phosphorylation mark in H3 is strengthened by the finding that MSK1, a kinase activated by growth factor and stress stimuli, also phosphorylates H3 in vitro.
H3 phosphorylation at serine 10 in conjunction with phosphorylation at serine 28, is also required for proper segregation and condensation of chromosomes during mitosis and meiosis.
ChIP results obtained with ab195407 directed against Histone H3 (acetyl K9, phospho S10).
ChIP assays were performed using chromatin from 1.5 million human osteosarcoma (U2OS) cells, ab195407 and optimized PCR primer sets for qPCR. A titration of the antibody consisting of 5 and 15 μl per ChIP experiment was analysed. IgG (5 μg/IP) was used as negative IP control. qPCR was performed with primers for the ALDOA (fructose-bisphosphate aldolase A) promoter and for the coding region of the myogenic differentiation gene (MYOD), a gene that is inactive at normal conditions. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
A Dot Blot analysis was performed to test the cross reactivity of ab195407 against Histone H3 (acetyl K9, phospho S10) peptide with peptides containing other modifications of histone H4 and H3 or unmodified histone H3 sequences. One hundred to 0.2 pmol of the peptide containing the respective histone modification were spotted on a membrane. The antibody was used at a dilution of 1/20000. The figure shows a high specificity of the antibody for the double modification.
Western blot - Anti-Histone H3 (acetyl K9, phospho S10) antibody - ChIP Grade (ab195407)
Anti-Histone H3 (acetyl K9, phospho S10) antibody - ChIP Grade (ab195407) at 1/250 dilution + HeLa histone extract at 15 µg
The antibody was diluted in TBS-Tween containing 5% skimmed milk.
Immunofluorescent analysis of HeLa cells labeling Histone H3 (acetyl K9, phospho S10) with ab195407 at 1/500 dilution.
Cells were fixed with 4% formaldehyde for 10 minutes and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with ab195407 (left) diluted 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.