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15002
AML1 (D33G6) XP® Rabbit mAb (PE Conjugate)
Antibody Conjugates

AML1 (D33G6) XP® Rabbit mAb (PE Conjugate) #15002

APPLICATIONS

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

Flow cytometric analysis of Jurkat cells using AML1 (D33G6) XP® Rabbit mAb (PE Conjugate) (green) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (red).

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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 antibodies. Protect from light. Do not freeze.

AML1 (D33G6) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total AML1 protein.

Species Reactivity:

Human, Monkey

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to amino acids at the amino terminus of human AML1 protein.

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated AML1 (D33G6) XP® Rabbit mAb #4336.

AML1 (also known as Runx1, CBFA2, and PEBP2αB) is a member of the core binding factor (CBF) family of transcription factors (1,2). It is required for normal development of all hematopoietic lineages (3-5). AML1 forms a heterodimeric DNA binding complex with its partner protein CBFβ and regulates the expression of cellular genes by binding to promoter and enhancer elements. AML1 is commonly translocated in hematopoietic cancers: chromosomal translocations include t(8;21) AML1-ETO, t(12;21) TEL-AML, and t(8;21) AML-M2 (6). Phosphorylation of AML1 on several potential serine and threonine sites, including Ser249, is thought to occur in an Erk-dependent manner (7,8).

  1. Wang, S. et al. (1993) Mol Cell Biol 13, 3324-39.
  2. Ogawa, E. et al. (1993) Proc Natl Acad Sci U S A 90, 6859-63.
  3. Okuda, T. et al. (1996) Cell 84, 321-30.
  4. Wang, Q. et al. (1996) Proc Natl Acad Sci U S A 93, 3444-9.
  5. North, T.E. et al. (2004) Stem Cells 22, 158-68.
  6. Blyth, K. et al. (2005) Nat Rev Cancer 5, 376-87.
  7. Tanaka, T. et al. (1996) Mol Cell Biol 16, 3967-79.
  8. Zhang, Y. et al. (2004) J Biol Chem 279, 53116-25.
Entrez-Gene Id
861
Swiss-Prot Acc.
Q01196
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
15002S
100 µl (50 tests)

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