The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Use at an assay dependent concentration.
This antibody gave a positive result in ELISA against the immunizing peptide (ab32016). It shows very weak reaction with the non-modified equivalent peptide (ab32100).This indicates that it is specific for the modified peptide. See figure below.
FunctionCore component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling.
Sequence similaritiesBelongs to the histone H2A family.
Post-translational modificationsThe chromatin-associated form is phosphorylated on Thr-121 during mitosis. Deiminated on Arg-4 in granulocytes upon calcium entry. Monoubiquitination of Lys-120 (H2AK119Ub) by RING1 and RNF2/RING2 complex gives a specific tag for epigenetic transcriptional repression and participates in X chromosome inactivation of female mammals. It is involved in the initiation of both imprinted and random X inactivation. Ubiquitinated H2A is enriched in inactive X chromosome chromatin. Ubiquitination of H2A functions downstream of methylation of 'Lys-27' of histone H3 (H3K27me). H2AK119Ub by RNF2/RING2 can also be induced by ultraviolet and may be involved in DNA repair. Following DNA double-strand breaks (DSBs), it is ubiquitinated through 'Lys-63' linkage of ubiquitin moieties by the E2 ligase UBE2N and the E3 ligases RNF8 and RNF168, leading to the recruitment of repair proteins to sites of DNA damage. Ubiquitination at Lys-14 and Lys-16 (H2AK13Ub and H2AK15Ub, respectively) in response to DNA damage is initiated by RNF168 that mediates monoubiquitination at these 2 sites, and 'Lys-63'-linked ubiquitin are then conjugated to monoubiquitin; RNF8 is able to extend 'Lys-63'-linked ubiquitin chains in vitro. H2AK119Ub and ionizing radiation-induced 'Lys-63'-linked ubiquitination (H2AK13Ub and H2AK15Ub) are distinct events. Phosphorylation on Ser-2 is enhanced during mitosis. Phosphorylation on Ser-2 by RPS6KA5/MSK1 directly represses transcription. Acetylation of H3 inhibits Ser-2 phosphorylation by RPS6KA5/MSK1. Symmetric dimethylation on Arg-4 by the PRDM1/PRMT5 complex may play a crucial role in the germ-cell lineage. Crotonylation (Kcr) is specifically present in male germ cells and marks testis-specific genes in post-meiotic cells, including X-linked genes that escape sex chromosome inactivation in haploid cells. Crotonylation marks active promoters and enhancers and confers resistance to transcriptional repressors. It is also associated with post-meiotically activated genes on autosomes.
IHC image of ab30819 staining Histone H2A (Hydroxy P26) in human colon formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab30819, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the negative control (shown on the inset).
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
This antibody gave a positive result in ELISA against the immunizing peptide (ab32016). It shows very weak reaction with the the non-modified equivalent peptide (ab32100). This indicates that it is specific for the modified peptide.
References for Anti-Histone H2A (hydroxy P26) antibody (ab30819)
This product has been referenced in:
Jagannathan R et al. AT1 receptor induced alterations in histone H2A reveal novel insights into GPCR control of chromatin remodeling. PLoS One5:e12552 (2010).
Read more (PubMed: 20838438) »