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 before commencing with IHC staining protocol.
Use a concentration of 1 µg/ml. Detects a band of approximately 16 kDa (predicted molecular weight: 14 kDa).
Use a concentration of 4 µg/ml. PubMed: 20693536
Use a concentration of 1 µg/ml.
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 H2B family.
Post-translational modificationsMonoubiquitination of Lys-121 by the RNF20/40 complex gives a specific tag for epigenetic transcriptional activation and is also prerequisite for histone H3 'Lys-4' and 'Lys-79' methylation. It also functions cooperatively with the FACT dimer to stimulate elongation by RNA polymerase II. Phosphorylated on Ser-15 by STK4/MST1 during apoptosis; which facilitates apoptotic chromatin condensation. Also phosphorylated on Ser-15 in response to DNA double strand breaks (DSBs), and in correlation with somatic hypermutation and immunoglobulin class-switch recombination.
Western blot - Histone H2B (acetyl K16) antibody (ab40977)
All lanes : Anti-Histone H2B (acetyl K16) antibody - ChIP Grade (ab40977) at 1 µg/ml
Lane 1 : HeLa Histone Preparation Nuclear Lysate Lane 2 : HeLa Histone Preparation Nuclear Lysate - Butyrated treated
Lane 3 : HeLa Histone Preparation Nuclear Lysate with Human Histone H2B (acetyl K16) peptide (ab41879) at 1 µg/ml Lane 4 : HeLa Histone Preparation Nuclear Lysate - Butyrated treated
with Human Histone H2B (acetyl K16) peptide (ab41879) at 1 µg/ml Lane 5 : HeLa Histone Preparation Nuclear Lysate with Human Histone H2B peptide (ab35793) at 1 µg/ml Lane 6 : HeLa Histone Preparation Nuclear Lysate - Butyrated treated
with Human Histone H2B peptide (ab35793) at 1 µg/ml
Lysates/proteins at 2.5 µg per lane.
Secondary HRP Conjugated Goat Anti-Rabbit IgG at 1/3000 dilution
ICC/IF image of ab40977 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab40977, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
IHC image of ab40977 staining in cervix formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. 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 ab40977, 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.
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.
Singh P et al. Coordinated allele-specific histone acetylation at the differentially methylated regions of imprinted genes. Nucleic Acids Res38:7974-90 (2010).
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