Anti-Histone H4 (di methyl K20) antibody - ChIP Grade (ab9052)
- Datasheet
- Specific References (24)
- Protocols
Overview
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Product nameAnti-Histone H4 (di methyl K20) antibody - ChIP Grade
See all Histone H4 primary antibodies -
DescriptionRabbit polyclonal to Histone H4 (di methyl K20) - ChIP Grade
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Host speciesRabbit
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Tested applicationsSuitable for: IP, WB, ChIP, ICC/IF, IHC-Pmore details
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Species reactivityReacts with: Mouse, Cow, Human, Schizosaccharomyces pombe, Toxoplasma gondii
Predicted to work with: Rat, a wide range of other species
Does not react with:
Saccharomyces cerevisiae -
Immunogen
Synthetic peptide within Saccharomyces cerevisiae Histone H4 aa 1-100 (di methyl K20) conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary.
(Peptide available asab26213) -
Positive control
- WB: Calf thymus histone preparation and HeLa whole cell extract. IHC-P: FFPE human pancreas adenocarcinoma tissue.
Properties
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FormLiquid
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Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
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Storage bufferPreservative: 0.02% Sodium Azide
Constituents: 1% BSA, PBS, pH 7.4 -
Concentration information loading... -
PurityImmunogen affinity purified
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ClonalityPolyclonal
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IsotypeIgG
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Research areas
Associated products
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ChIP Related Products
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Compatible Secondaries
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Related Products
Applications
Our Abpromise guarantee covers the use of ab9052 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Abreviews | Notes |
|---|---|---|
| IP | Use at an assay dependent concentration. | |
| WB | 1/2000. Detects a band of approximately 11 kDa. | |
| ChIP | Use at an assay dependent concentration. PubMed: 20829797 | |
| ICC/IF | Use a concentration of 1 µg/ml. | |
| IHC-P | Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. |
Target
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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.
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Sequence similaritiesBelongs to the histone H4 family.
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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. -
Cellular localizationNucleus. Chromosome.
- Information by UniProt
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Database links
- Entrez Gene: 121504 Human
- Entrez Gene: 554313 Human
- Entrez Gene: 8294 Human
- Entrez Gene: 8359 Human
- Entrez Gene: 8360 Human
- Entrez Gene: 8361 Human
- Entrez Gene: 8362 Human
- Entrez Gene: 8363 Human
see all -
Alternative names
- H4_HUMAN antibody
- H4F2 antibody
- H4FN antibody
see all
Images
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Immunocytochemistry/ Immunofluorescence - Anti-Histone H4 (di methyl K20) antibody - ChIP Grade (ab9052)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.
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Immunoprecipitation - Anti-Histone H4 (di methyl K20) antibody - ChIP Grade (ab9052)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). -
Western blot - Anti-Histone H4 (di methyl K20) antibody - ChIP Grade (ab9052)This image is courtesy of Steve Sanders, Tony Kouzarides lab, University of CambridgeAll 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. -
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.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 (di methyl K20) antibody - ChIP Grade (ab9052)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.
Protocols
References
This product has been referenced in:
- Gao Y & Ge W The histone methyltransferase DOT1L inhibits osteoclastogenesis and protects against osteoporosis. Cell Death Dis 9:33 (2018). Read more (PubMed: 29348610) »
- Pellegrino S et al. Replication-Coupled Dilution of H4K20me2 Guides 53BP1 to Pre-replicative Chromatin. Cell Rep 19:1819-1831 (2017). WB, ICC/IF ; Human . Read more (PubMed: 28564601) »
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