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54236
FoxM1 (D3F2B) Rabbit mAb (Flow Formulated)
Primary Antibodies

FoxM1 (D3F2B) Rabbit mAb (Flow Formulated) #54236

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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 (Flow Formulated) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

To Purchase # 54236S
Product # Size Price
54236S
100 µl $ 260

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa)
Isotype Rabbit IgG

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Usage Information

Application Dilutions
Flow Cytometry 1:100

Storage:

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Protocol

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Flow Cytometry, Methanol Permeabilization Protocol for Rabbit 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.
  5. Recommended Anti-Rabbit secondary antibodies::
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) #8889
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #79408

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.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in Antibody Dilution Buffer at the recommended dilution).
  7. Incubate for 30 min at room temperature. Protect from light.
  8. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  9. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised August 2019

Protocol Id: 404

Specificity / Sensitivity

FoxM1 (D3F2B) Rabbit mAb (Flow Formulated) 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.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.