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34113
MeCP2 (D4F3) XP® Rabbit mAb (PE Conjugate)

MeCP2 (D4F3) XP® Rabbit mAb (PE Conjugate) #34113

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Mk Endogenous Rabbit IgG
Flow Cytometry

Flow cytometric analysis of SH-SY5Y cells using using MeCP2 (D4F3) XP® Rabbit mAb (PE Conjugate) (solid line) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed line).

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Application Dilutions
Flow Cytometry 1:50
Storage:

Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

MeCP2 (D4F3) XP® Rabbit mAb (PE Conjugate) detects endogenous levels of MeCP2 (both isoforms A and B). This antibody does not cross-react with other MBD proteins.

Species Reactivity:

Human, Mouse, Rat, Monkey

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the carboxy terminus of human MeCP2.

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated MeCP2 (D4F3) XP® Rabbit mAb #3456.

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.
Entrez-Gene Id
4204
Swiss-Prot Acc.
P51608
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

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To Purchase # 34113S

Product Number Size Price
34113S 100 µl (50 tests) $341.00.0
Quantity Subtotal
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