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71517
DNMT3B (D7O7O) Rabbit mAb (PE Conjugate)

DNMT3B (D7O7O) Rabbit mAb (PE Conjugate) #71517

APPLICATIONS

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

Flow cytometric analysis of HCT 116 cells (blue) and NCCIT cells (green) using DNMT3B (D7O7O) Rabbit mAb (PE Conjugate) (solid lines) or a Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).

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Flow Cytometry Triton™ X-100 Permeabilization Protocol - Conjugated Antibody

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. Adjust pH to 8.0.
  2. Formaldehyde (methanol free).
  3. Incubation Buffer (1X PBS / 0.5% BSA): Dissolve 0.5 g bovine serum albumin (BSA) in 100 mL 1X PBS. Store at 4°C.
  4. Permeabilization Buffer (1X PBS / 0.3% Triton™ X-100 / 0.5% BSA): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml Incubation Buffer.

B. Fixation and Permabilization

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 briefly in 0.5-1 ml PBS. Add formaldehyde to a final concentration of 2-4% formaldehyde.
  3. Fix for 15 minutes at room temperature (20-25 °C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container.
  5. Resuspend cells in 1 ml Permeabilization Buffer.
  6. Incubate for 10 minutes at room temperature.
  7. Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.

C. Staining Using Conjugated Primary Antibody

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells (suggested range of 0.5x106 to 1x106 cells).
  2. Centrifuge cells and discard supernatant.
  3. Resuspend cells in 100 µl of diluted conjugated primary antibody (prepared in Incubation Buffer at the recommended dilution).
  4. Incubate for 30-60 min at room temperature (20-25 °C). Protect from light.
  5. Wash by centrifugation in 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section D).

D. Optional DNA Dye

  1. Resuspend cells in 0.1-0.5 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 January 2017

revised May 2018

Protocol Id: 1344

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.

DNMT3B (D7O7O) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total DNMT3B protein. This antibody also detects a non-specific protein of approximately 65 kDa in multiple cell lines. Based on sequence homology, this antibody should recognize all isoforms of DNMT3B. This antibody shows low sensitivity in IF-IC, where it only detects DNMT3B in high expressing cells. However, this clone detects DNMT3B in both high and low expressing cells by western blot.

Species Reactivity:

Human

Monoclonal antibody is produced by immunizing animals with recombinant protein surrounding Ala395 of human DNMT3B protein.

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 DNMT3B (D7O7O) Rabbit mAb #67259.

Methylation of DNA at cytosine residues in mammalian cells is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting and development (1,2). Three families of mammalian DNA methyltransferases have been identified: DNMT1, DNMT2 and DNMT3 (1,2). DNMT1 is constitutively expressed in proliferating cells and functions as a maintenance methyltransferase, transferring proper methylation patterns to newly synthesized DNA during replication. DNMT3A and DNMT3B are strongly expressed in embryonic stem cells with reduced expression in adult somatic tissues. DNMT3A and DNMT3B function as de novo methyltransferases that methylate previously unmethylated regions of DNA. DNMT2 is expressed at low levels in adult somatic tissues and its inactivation affects neither de novo nor maintenance DNA methylation. DNMT1, DNMT3A and DNMT3B together form a protein complex that interacts with histone deacetylases (HDAC1, HDAC2, Sin3A), transcriptional repressor proteins (RB, TAZ-1) and heterochromatin proteins (HP1, SUV39H1), to maintain proper levels of DNA methylation and facilitate gene silencing (3-8). Improper DNA methylation contributes to diseased states such as cancer (1,2). Hypermethylation of promoter CpG islands within tumor suppressor genes correlates with gene silencing and the development of cancer. In addition, hypomethylation of bulk genomic DNA correlates with and may contribute to the onset of cancer. DNMT1, DNMT3A and DNMT3B are over-expressed in many cancers, including acute and chronic myelogenous leukemias, in addition to colon, breast and stomach carcinomas (9-12).

  1. Hermann, A. et al. (2004) Cell Mol Life Sci 61, 2571-87.
  2. Turek-Plewa, J. and Jagodziński, P.P. (2005) Cell Mol Biol Lett 10, 631-47.
  3. Kim, G.D. et al. (2002) EMBO J. 21, 4183-4195.
  4. Fuks, F. et al. (2001) EMBO J. 20, 2536-2544.
  5. Geiman, T.M. et al. (2004) Biochem. Biophys. Res. Commun. 318, 544-555.
  6. Rountree, M.R. et al. (2000) Nat. Genet. 25, 269-277.
  7. Pradhan, S. and Kim, G.D. (2002) EMBO J. 21, 779-788.
  8. Fuks, F. et al. (2003) Nucleic Acids Res. 31, 2305-2312.
  9. Mizuno, S. et al. (2001) Blood 97, 1172-1179.
  10. Robertson, K.D. et al. (1999) Nucleic Acids Res. 27, 2291-2298.
  11. Xie, S. et al. (1999) Gene 236, 87-95.
  12. Kanai, Y. et al. (2001) Int. J. Cancer 91, 205-212.
Entrez-Gene Id
1789
Swiss-Prot Acc.
Q9UBC3
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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Product # Size Price
71517S
100 µl (50 tests) $ 299.0

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