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73288
c-Myb (D1B9E) Rabbit mAb (PE Conjugate)

c-Myb (D1B9E) Rabbit mAb (PE Conjugate) #73288

APPLICATIONS

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

Flow cytometric analysis of 293T cells (blue) or Jurkat cells (green) using c-Myb (D1B9E) Rabbit mAb (PE Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).

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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.

c-Myb (D1B9E) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total c-Myb protein.

Species Reactivity:

Human

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the carboxy terminus of human c-Myb 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 c-Myb (D1B9E) Rabbit mAb #59995.

c-Myb is a transcriptional activator that specifically recognizes the sequence 5'-YAAC[GT]G-3'. It is expressed in hematopoietic progenitor cells where it plays an important role in the control of proliferation and differentiation (1-3). c-Myb is required for transcription of genes involved in self-renewal of intestinal stem cells. Importantly, c-Myb regulates expression of Lgr5, a protein expressed in putative intestinal stem cells that give rise to all cell lineages of small intestinal crypts (4). c-Myb is reported to be expressed in colon crypt cells and in human colorectal cancer lines (5,6). Research has shown that c-Myb gene translocations and copy number alterations are found in several leukemias, breast cancer, and other solid tumors (7,8).

  1. Lin, H.H. et al. (1996) Curr Top Microbiol Immunol 211, 79-87.
  2. Mucenski, M.L. et al. (1991) Cell 65, 677-89.
  3. Badiani, P. et al. (1994) Genes Dev 8, 770-82.
  4. Cheasley, D. et al. (2011) Stem Cells 29, 2042-50.
  5. Thompson, M.A. et al. (1998) Cancer Res 58, 5168-75.
  6. Wilkins, H.R. et al. (2010) Tumour Biol 31, 16-22.
  7. Ramsay, R.G. and Gonda, T.J. (2008) Nat Rev Cancer 8, 523-34.
  8. Stenman, G. et al. (2010) Cell Cycle 9, 2986-95.
Entrez-Gene Id
4602
Swiss-Prot Acc.
P10242
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 # 73288S

Product Number Size Price
73288S 100 µl (50 tests) $299.00.0
Quantity Subtotal
$0.00

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