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92319
FoxM1 (D3F2B) Rabbit mAb (PE Conjugate)
Antibody Conjugates

Monoclonal Antibody - FoxM1 (D3F2B) Rabbit mAb (PE Conjugate), UniProt ID Q08050, Entrez ID 2305 #92319

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Flow cytometric analysis of HeLa cells, untreated (blue) or treated with Aphidicolin (2 μM, 24 hr; green), using FoxM1 (D3F2B) Rabbit mAb (PE Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).

To Purchase # 92319S

Supporting Data

REACTIVITY H
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 FoxM1 (D3F2B) Rabbit mAb #20459.

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 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

Specificity / Sensitivity

FoxM1 (D3F2B) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total FoxM1 protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val699 of human FoxM1 protein.

Background

Forkhead box M1 (FoxM1) is a forkhead box family transcription factor that regulates a number of genes throughout the cell cycle to help control DNA replication, mitosis, and cell proliferation. FoxM1 expression increases during G1 and S and reaches maximum levels in G2/M (1-3). Nuclear translocation occurs just before entry into G2/M and is associated with FoxM1 phosphorylation (4). Phosphorylation of FoxM1 by MAPK (Ser331, Ser704), Cyclin/Cdk (Ser4, Ser35, Thr600, Thr611, Thr620, Thr627, Ser638), Plk1 (Ser715, Ser724), and Chk2 (Ser376) stabilizes and activates FoxM1 (4-8). Forkhead box M1 is expressed in all embryonic tissues but is restricted to proliferating tissues in adults (9). Research studies show that FoxM1 expression is negatively regulated by p53 (10,11). Upregulation of FoxM1 is associated with many human cancers, including prostate, breast, lung, ovary, colon, pancreas, stomach, bladder, liver, and kidney, and may be associated with p53 mutations in some tumors (11,12). As a result, FoxM1 inhibitors have become a topic of interest for potential cancer therapy (13).

  1. Wang, I.C. et al. (2005) Mol Cell Biol 25, 10875-94.
  2. Leung, T.W. et al. (2001) FEBS Lett 507, 59-66.
  3. Wang, X. et al. (2002) Proc Natl Acad Sci U S A 99, 16881-6.
  4. Ma, R.Y. et al. (2005) J Cell Sci 118, 795-806.
  5. Laoukili, J. et al. (2008) Mol Cell Biol 28, 3076-87.
  6. Fu, Z. et al. (2008) Nat Cell Biol 10, 1076-82.
  7. Tan, Y. et al. (2007) Mol Cell Biol 27, 1007-16.
  8. Anders, L. et al. (2011) Cancer Cell 20, 620-34.
  9. Ye, H. et al. (1997) Mol Cell Biol 17, 1626-41.
  10. Barsotti, A.M. and Prives, C. (2009) Oncogene 28, 4295-305.
  11. Pandit, B. et al. (2009) Cell Cycle 8, 3425-7.
  12. Pilarsky, C. et al. (2004) Neoplasia 6, 744-50.
  13. Gartel, A.L. (2008) Expert Opin Ther Targets 12, 663-5.

Pathways & Proteins

Explore pathways + proteins related to this product.

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

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