Revision 3

#99169Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, ChIP, ChIP-seq, C&R

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

32, 34

Source/Isotype:

Rabbit IgG

UniProt ID:

#O95983

Entrez-Gene Id:

53615

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq 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
Chromatin IP 1:50
Chromatin IP-seq 1:50
CUT&RUN 1:50

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

MBD3 (D1B8F) Rabbit mAb recognizes endogenous levels of total MBD3 protein.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val81 of human MBD3 protein.

Background

Methyl-CpG-binding protein 2 (MeCP2) is the founding member of a family of methyl-CpG-binding domain (MBD) proteins that also includes MBD1, MBD2, MBD3, MBD4, MBD5, and MBD6 (1-3). Apart from MBD3, these proteins bind methylated cytosine residues in the context of the di-nucleotide 5´-CG-3´ to establish and maintain regions of transcriptionally inactive chromatin by recruiting a variety of co-repressor proteins (2). MeCP2 recruits histone deacetylases HDAC1 and HDAC2, and the DNA methyltransferase DNMT1 (4-6). MBD1 couples transcriptional silencing to DNA replication and interacts with the histone methyltransferases ESET and SUV39H1 (7,8). MBD2 and MBD3 co-purify as part of the NuRD (nucleosome remodeling and histone de-acetylation) co-repressor complex, which contains the chromatin remodeling ATPase Mi-2, HDAC1, and HDAC2 (9,10). MBD5 and MBD6 have recently been identified and little is known regarding their protein interactions. MBD proteins are associated with cancer and other diseases; MBD4 is best characterized for its role in DNA repair and MBD2 has been linked to intestinal cancer (11,12). Mutations in the MeCP2 gene cause the neurologic developmental disorder Rett Syndrome (13). MeCP2 protein levels are high in neurons, where it plays a critical role in multiple synaptic processes (14). In response to various physiological stimuli, MeCP2 is phosphorylated on Ser421 and regulates the expression of genes controlling dendritic patterning and spine morphogenesis (14). Disruption of this process in individuals with altered MeCP2 may cause the pathological changes seen in Rett Syndrome.

  1. Clouaire, T. and Stancheva, I. (2008) Cell Mol Life Sci 65, 1509-22.
  2. Hendrich, B. and Bird, A. (1998) Mol Cell Biol 18, 6538-47.
  3. Roloff, T.C. et al. (2003) BMC Genomics 4, 1.
  4. Nan, X. et al. (1998) Nature 393, 386-9.
  5. Jones, P.L. et al. (1998) Nat Genet 19, 187-91.
  6. Fuks, F. et al. (2003) J Biol Chem 278, 4035-40.
  7. Sarraf, S.A. and Stancheva, I. (2004) Mol Cell 15, 595-605.
  8. Fujita, N. et al. (2003) J Biol Chem 278, 24132-8.
  9. Zhang, Y. et al. (1999) Genes Dev 13, 1924-35.
  10. Wade, P.A. et al. (1999) Nat Genet 23, 62-6.
  11. Hendrich, B. et al. (1999) Nature 401, 301-4.
  12. Sansom, O.J. et al. (2003) Nat Genet 34, 145-7.
  13. Miltenberger-Miltenyi, G. and Laccone, F. (2003) Hum Mutat 22, 107-15.
  14. Zhou, Z. et al. (2006) Neuron 52, 255-69.

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

WB: Western Blotting ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq 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 All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SimpleChIP 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 3
#99169

MBD3 (D1B8F) Rabbit mAb

Western Blotting Image 1: MBD3 (D1B8F) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using MBD3 (D1B8F) Rabbit mAb.
Chromatin Immunoprecipitation Image 1: MBD3 (D1B8F) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from K562 cells and MBD3 (D1B8F) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across ZC3H11A gene.
Chromatin Immunoprecipitation Image 2: MBD3 (D1B8F) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from K562 cells and MBD3 (D1B8F) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across chromosome 1 (upper), including ZC3H11A (lower).
Chromatin Immunoprecipitation Image 3: MBD3 (D1B8F) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from K562 cells and either MBD3 (D1B8F) 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 GAPDH Exon 1 Primers #5516, human BCR exon 1 primers, and SimpleChIP® Human Nanog Promoter Primers #95064. 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 and RUN Image 1: MBD3 (D1B8F) Rabbit mAb Expand Image
CUT&RUN was performed with K562 cells and MBD3 (D1B8F) 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 CTPS1 gene, a known target gene of MBD3 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: MBD3 (D1B8F) Rabbit mAb Expand Image
CUT&RUN was performed with K562 cells and MBD3 (D1B8F) 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 chromosome #1 (upper), including CTPS1 (lower), a known target gene of MBD3 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: MBD3 (D1B8F) Rabbit mAb Expand Image
CUT&RUN was performed with K562 cells and either MBD3 (D1B8F) 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 CTPS1 promoter primers and human PANTR1 promoter primers. 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.