Revision 3
#11974
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For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:
W, IP, ChIP, C&R
Reactivity:
H M R Mk
Sensitivity:
Endogenous
MW (kDa):
120
Source/Isotype:
Rabbit IgG
UniProt ID:
#Q5VTR2
Entrez-Gene Id:
56254
Product Usage Information
For optimal ChIP results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.
The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:200 |
Chromatin IP | 1:50 |
CUT&RUN | 1:50 |
Storage
Specificity/Sensitivity
RNF20 (D6E10) XP® Rabbit mAb recognizes endogenous levels of total RNF20 protein. This antibody recognizes a second band at 80 kDa in mouse lysates, corresponding to mouse RNF20 isoform 2. This antibody does not cross-react with RNF40 protein.
Species predicted to react based on 100% sequence homology
Hamster, Dog, Pig, Horse, Guinea Pig
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly516 of human RNF20 protein.
Background
In mammalian cells, the significance of histone H2B ubiquitination in chromatin epigenetics came from the identification of the budding yeast protein Bre1 (1,2). Together with the ubiquitin-conjugating enzyme Rad6, Bre1 serves as the E3 ligase in the monoubiquitination of the yeast histone H2B within transcribed regions of chromatin (1-3). Subsequently, the mammalian orthologs of yeast Bre1, RNF20 and RNF40, were identified (4,5). These two proteins form a tight heterodimer that acts as the major E3 ligase responsible for histone H2B monoubiquitination at Lys120 in mammalian cells, a modification linked to RNA Pol II-dependent transcription elongation in undamaged cells. Researchers have shown that DNA double-strand breaks (DSBs) are also capable of inducing monoubiquitination of H2B. This process depends upon the recruitment to DSB sites, as well as ATM-dependent phosphorylation of the RNF20-RNF40 heterodimer, thus highlighting a role for this E3 ligase in DSB repair pathways (6). Indeed, investigators have shown that loss of RNF20-RNF40 function promotes replication stress and chromosomal instability, which may constitute an early step in malignant transformation that precedes cell invasion (7).
Background References
- Wood, A. et al. (2003) Mol Cell 11, 267-74.
- Hwang, W.W. et al. (2003) Mol Cell 11, 261-6.
- Kao, C.F. et al. (2004) Genes Dev 18, 184-95.
- Kim, J. et al. (2005) Mol Cell 20, 759-70.
- Zhu, B. et al. (2005) Mol Cell 20, 601-11.
- Moyal, L. et al. (2011) Mol Cell 41, 529-42.
- Chernikova, S.B. et al. (2012) Cancer Res, Epub ahead of print.
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 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
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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 3
#11974
RNF20 (D6E10) XP® Rabbit mAb
Western blot analysis of extracts from various cell lines using RNF20 (D6E10) XP® Rabbit mAb.
Chromatin immunoprecipitations were performed with cross-linked chromatin fromHeLa cells and either RNF20 (D6E10) XP® Rabbit mAb 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® Human γ-Actin Promoter Primers #5037, SimpleChIP® Human GAPDH Promoter Primers #4471, and SimpleChIP® Human α Satellite Repeat Primers #4486. 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 RNF20 (D6E10) 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 across TAF6L gene, a known target gene of RNF20 (see additional figure containing CUT&RUN-qPCR data).
Revision 3
#11974
RNF20 (D6E10) XP® Rabbit mAb
CUT&RUN was performed with HeLa cells and RNF20 (D6E10) 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 TAF6L (upper) and HOXA (lower) gene regions, known target genes of RNF20 (see additional figure containing CUT&RUN-qPCR data).
CUT&RUN was performed with HeLa cells and either RNF20 (D6E10) 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 human TAF6L promoter primers and SimpleChIP® Human α Satellite Repeat Primers #4486. 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.