The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 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 (ab22398). For the following peptides it gave following results: the non-modified equivalent peptide (ab13186) - negative result, Histone H2A (symmetric di methyl R3)peptide(ab22399) - negative result. This indicates that it is specific for the Histone H2A (asymmetric di methyl R3) peptide. See figure below. Not yet tested in other applications. Optimal dilutions/concentrations should be determined by the end user.
Core 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.
Belongs to the histone H2A family.
The chromatin-associated form is phosphorylated on Thr-121 during mitosis. Deiminated on Arg-4 in granulocytes upon calcium entry. Monoubiquitination of Lys-120 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. Monoubiquitination of Lys-120 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. Monoubiquitination and ionizing radiation-induced 'Lys-63'-linked ubiquitination 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.
IHC image of ab21574 staining Histone H2A (asymmetric di methyl R3) 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 ab21574, 1µg/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.
ELISA - Histone H2A (asymmetric di methyl R3) antibody (ab21574)
This antibody gave a positive result in ELISA against the immunizing peptide ab22398 (blue line). For the following peptides it gave following results: Histone H2A (symmetric di methyl R3) peptide ab22399 (pink line) - negative result, the non-modified equivalent peptide ab13186 (yellow line) - negative result. This indicates that it is specific for the Histone H2A (asymmetric di methyl R3) peptide.