The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
1/2000. Detects a band of approximately 11 kDa.
Use at an assay dependent concentration. PubMed: 20829797
Use a concentration of 1 µg/ml.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
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 H4 family.
Post-translational modificationsAcetylation at Lys-6 (H4K5ac), Lys-9 (H4K8ac), Lys-13 (H4K12ac) and Lys-17 (H4K16ac) occurs in coding regions of the genome but not in heterochromatin. Citrullination at Arg-4 (H4R3ci) by PADI4 impairs methylation. Monomethylation and asymmetric dimethylation at Arg-4 (H4R3me1 and H4R3me2a, respectively) by PRMT1 favors acetylation at Lys-9 (H4K8ac) and Lys-13 (H4K12ac). Demethylation is performed by JMJD6. Symmetric dimethylation on Arg-4 (H4R3me2s) by the PRDM1/PRMT5 complex may play a crucial role in the germ-cell lineage. Monomethylated, dimethylated or trimethylated at Lys-21 (H4K20me1, H4K20me2, H4K20me3). Monomethylation is performed by SET8. Trimethylation is performed by SUV420H1 and SUV420H2 and induces gene silencing. Phosphorylated by PAK2 at Ser-48 (H4S47ph). This phosphorylation increases the association of H3.3-H4 with the histone chaperone HIRA, thus promoting nucleosome assembly of H3.3-H4 and inhibiting nucleosome assembly of H3.1-H4. Ubiquitinated by the CUL4-DDB-RBX1 complex in response to ultraviolet irradiation. This may weaken the interaction between histones and DNA and facilitate DNA accessibility to repair proteins. Monoubiquitinated at Lys-92 of histone H4 (H4K91ub1) in response to DNA damage. The exact role of H4K91ub1 in DNA damage response is still unclear but it may function as a licensing signal for additional histone H4 post-translational modifications such as H4 Lys-21 methylation (H4K20me). Sumoylated, which is associated with transcriptional repression. 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.
ICC/IF image of ab9052 stained HeLa cells. The cells were 100% methanol fixed (5 min) then permeabilised using 0.1% PBS-Triton and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to further permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab9052 at 1µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
Western blot - Anti-Histone H4 (di methyl K20) antibody - ChIP Grade (ab9052)This image is courtesy of Steve Sanders, Tony Kouzarides lab, University of Cambridge
All lanes : Anti-Histone H4 (di methyl K20) antibody - ChIP Grade (ab9052) at 1/2000 dilution
Lane 1 : yH4 17-24 peptide Lane 2 : Unmodified yH4 17-24 peptide Lane 3 : Mono methyl K20 yH4 17-24 peptide Lane 4 : Di methyl K20 yH4 17-24 peptide Lane 5 : Tri methyl K20 yH4 17-24 peptide
Blocking peptides at 1 µg/ml per lane.
Performed under reducing conditions.
This image is courtesy of Steve Sanders, Tony Kouzarides lab, University of Cambridge
Western blot - Anti-Histone H4 (di methyl K20) antibody - ChIP Grade (ab9052)
All lanes : Upper blot - Histone H4 antibody (gift of A. Verreault)
Lower blot - Rabbit polyclonal to Histone H4 di methyl K20 (ab9052) at 1/2000
Lane 1 : S.cerevisiae extract Lane 2 : S.pombe extract Lane 3 : S.pombe rH4 Lane 4 : Drosophila rH4 Lane 5 : Histones (Roche)
Di methylation at K20 is seen in S. pombe and in calf thymus (Roche). S. cerevisiae lacks K20 (di) methylation.
Histone H4 (di methyl K20) was immunoprecipitated using 0.5mg HeLa whole cell extract, 5µg of Rabbit polyclonal to Histone H4 (di methyl K20) and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab9052. Secondary: Clean blot (HRP conjugate) at 1/1000 dilution. Band: 14kDa: Histone H4 (di methyl K20).
IHC image of ab9052 staining in human pancreas adenocarcinoma 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 ab9052, 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.
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.
Svensson JP et al. A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin. Genome Res25:872-83 (2015).
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