Revision 5
#8676
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For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:
W, IP, IF-IC, ChIP, ChIP-seq, C&R
Reactivity:
H M R Mk
Sensitivity:
Endogenous
MW (kDa):
25
Source/Isotype:
Rabbit IgG
UniProt ID:
#P83916
Entrez-Gene Id:
10951
Product Usage Information
The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:50 |
Immunofluorescence (Immunocytochemistry) | 1:800 |
Chromatin IP | 1:50 |
Chromatin IP-seq | 1:50 |
CUT&RUN | 1:50 |
Storage
Specificity/Sensitivity
HP1β (D2F2) XP® Rabbit mAb recognizes endogenous levels of total HP1β protein. This antibody does not cross-react with other HP1 proteins, including HP1α and HP1γ.
Species predicted to react based on 100% sequence homology
Hamster, Bovine, Guinea Pig
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gln69 of human HP1β protein.
Background
Heterochromatin protein 1 (HP1) is a family of heterochromatic adaptor molecules involved in both gene silencing and higher order chromatin structure (1). All three HP1 family members (α, β, and γ) are primarily associated with centromeric heterochromatin; however, HP1β and γ also localize to euchromatic sites in the genome (2,3). HP1 proteins are approximately 25 kDa in size and contain a conserved amino-terminal chromodomain, followed by a variable hinge region and a conserved carboxy-terminal chromoshadow domain. The chromodomain facilitates binding to histone H3 tri-methylated at Lys9, a histone "mark" closely associated with centromeric heterochromatin (4,5). The variable hinge region binds both RNA and DNA in a sequence-independent manner (6). The chromoshadow domain mediates the dimerization of HP1 proteins, in addition to binding multiple proteins implicated in gene silencing and heterochromatin formation, including the SUV39H histone methyltransferase, the DNMT1 and DNMT3a DNA methyltransferases, and the p150 subunit of chromatin-assembly factor-1 (CAF1) (7-9). In addition to contributing to heterochromatin formation and propagation, HP1 and SUV39H are also found complexed with retinoblastoma (Rb) and E2F6 proteins, both of which function to repress euchromatic gene transcription in quiescent cells (10,11). HP1 proteins are subject to multiple types of post-translational modifications, including phosphorylation, acetylation, methylation, ubiquitination, and sumoylation, suggesting multiple means of regulation (12-14).
Background References
- Maison, C. and Almouzni, G. (2004) Nat. Rev. Mol. Cell Biol. 5, 296-304.
- Minc, E. et al. (2000) Cytogenet. Cell Genet. 90, 279-284.
- Nielsen, A.L. et al. (2001) Mol. Cell 7, 729-739.
- Lachner, M. et al. (2001) Nature 410, 116-120.
- Bannister, A.J. et al. (2001) Nature 410, 120-124.
- Muchardt, C. et al. (2002) EMBO Rep. 3, 975-981.
- Yamamoto, K. and Sonoda, M. (2003) Biochem. Biophys. Res. Commun. 301, 287-292.
- Fuks, F. et al. (2003) Nucleic Acids Res. 31, 2305-2312.
- Murzina, N. et al. (1999) Mol. Cell 4, 529-540.
- Nielsen, S.J. et al. (2001) Nature 412, 561-565.
- Ogawa, H. et al. (2002) Science 296, 1132-1136.
- Minc, E. et al. (1999) Chromosoma 108, 220-234.
- Zhao, T. et al. (2001) J. Biol. Chem. 276, 9512-9518.
- Lomberk, G. et al. (2006) Nat. Cell Biol. 8, 407-415.
Species Reactivity
Species reactivity is determined by testing in at least one approved application (e.g., western blot).
Western Blot Buffer
IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
Applications Key
W: Western Blotting IP: Immunoprecipitation IF-IC: Immunofluorescence (Immunocytochemistry) ChIP: Chromatin IP C&R: CUT&RUN
Cross-Reactivity Key
H: Human M: Mouse R: Rat Hm: Hamster Mk: Monkey Vir: Virus Mi: Mink C: Chicken Dm: D. melanogaster X: Xenopus Z: Zebrafish B: Bovine Dg: Dog Pg: Pig Sc: S. cerevisiae Ce: C. elegans Hr: Horse GP: Guinea Pig Rab: Rabbit G: Goat All: All Species Expected
Trademarks and Patents
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.
Limited Uses
Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.
Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.
Revision 5
Western blot analysis of extracts from various cell lines using HP1β (D2F2) XP® Rabbit mAb.
Confocal immunofluorescent analysis of C2C12 cells using HP1β (D2F2) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
Chromatin immunoprecipitations were performed with cross-linked chromatin from Hela cells and either HP1β (D2F2) XP® Rabbit mAb or Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb #13969, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. HP1β and H3K9me3 are known to associate with each other on chromatin. The figure shows binding of both HP1β and H3K9me3 across ZNF genes, known target genes of both HP1β and H3K9me3.
Revision 5
Chromatin immunoprecipitations were performed with cross-linked chromatin from mES cells and either HP1β (D2F2) XP® Rabbit mAb #8676 or Normal Rabbit IgG #2729, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Mouse MEST Intron 1 Primers #12870, SimpleChIP® Mouse MEST Promoter Primers #12928, mouse Intracisternal A-Particle (IAP) LTR promoter primers, and mouse primers specific to a non-genic, non-repetitive region. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
CUT&RUN was performed with HeLa cells and HP1β (D2F2) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figure shows binding upstream of RAB31 gene.
CUT&RUN was performed with HeLa cells and HP1β (D2F2) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across RAB31 (upper) and ZNF529 (lower).
Revision 5
CUT&RUN was performed with HeLa cells and either HP1β (D2F2) XP® Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human α Satellite Repeat Primers #4486 and SimpleChIP® Human ZNF335 Promoter Primers #25946. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.