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

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

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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).

To Purchase # 34113S
Product # Size Price
34113S
100 µl  (50 tests) $ 348

Supporting Data

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

Product Description

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.

Product Usage Information

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.

Protocol

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Flow Cytometry, Methanol Permeabilization Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.

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

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. 100% Methanol (#13604): Chill before use
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

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

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight at 4°C in 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. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  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 primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised August 2019

Protocol Id: 407

Specificity / Sensitivity

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

Source / Purification

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

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.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.

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