The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/2000. Customers have successfuly used ab17351 to detect Histone H2B di-methyl K5. For further details please see Abreview.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use at an assay dependent concentration. This antibody gave a positive result in ELISA against the immunizing peptide (ab26342). It gave a negative result in ELISA against the non-modified equivalent peptide (ab13212). This indicates that it is specific for the modified peptide.
See figure below.
Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 14 kDa).
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.
The nucleosome is a histone octamer containing two molecules each of H2A, H2B, H3 and H4 assembled in one H3-H4 heterotetramer and two H2A-H2B heterodimers. The octamer wraps approximately 147 bp of DNA.
Monoubiquitination at Lys-35 (H2BK34Ub) by the MSL1/MSL2 dimer is required for histone H3 'Lys-4' (H3K4me) and 'Lys-79' (H3K79me) methylation and transcription activation at specific gene loci, such as HOXA9 and MEIS1 loci. Similarly, monoubiquitination at Lys-121 (H2BK120Ub) 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. H2BK120Ub also acts as a regulator of mRNA splicing: deubiquitination by USP49 is required for efficient cotranscriptional splicing of a large set of exons.
Phosphorylation at Ser-37 (H2BS36ph) by AMPK in response to stress promotes transcription. Phosphorylated on Ser-15 (H2BS14ph) 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.
GlcNAcylation at Ser-113 promotes monoubiquitination of Lys-121. It fluctuates in response to extracellular glucose, and associates with transcribed genes.
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.
This antibody gave a positive result in ELISA against the immunizing peptide ab26342 (red line). It gave a negative result in ELISA against the non-modified equivalent peptide ab13212 (green line). This indicates that it is specific for the modified peptide.
IHC image of Histone H2B (di methyl K5) staining in human breast carcinoma FFPE section, performed on a 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 ab17351, 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.
Immunocytochemistry/ Immunofluorescence - Anti-Histone H2B (di methyl K5) antibody (ab17351)Image courtesy of an Abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada
ab17351 (1/2000) staining Histone H2B (di methyl K5) in HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilised using 0.5% Triton X100 and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.
ICC/IF image of ab17351 stained HeLa cells. The cells were 100% methanol fixed (5 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab17351, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to 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). This antibody also gave a positive IF result in 4%PFA and 100% methanol fixed Hek293, HepG2 and MCF7 cells. However, this Fast-Track antibody is not yet fully characterised. This image represents inconclusive preliminary data.