Buy Three, Get the Fourth Free! | Start Saving >>
54236
FoxM1 (D3F2B) Rabbit mAb (Flow Formulated)

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

APPLICATIONS

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

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.

Learn more about how we get our images.

Flow Cytometry, Methanol Permeabilization Protocol for Rabbit Antibodies

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.
  5. Recommended Anti-Rabbit secondary antibodies::

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

NOTE: Count cells using a hemocytometer or alternative method.

  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 primary antibody (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in incubation buffer at recommended dilution).
  7. Incubate for 30 min at room temperature.
  8. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  9. 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: 404

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.

FoxM1 (D3F2B) Rabbit mAb (Flow Formulated) recognizes endogenous levels of total FoxM1 protein.

Species Reactivity:

Human

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

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.
Entrez-Gene Id
2305
Swiss-Prot Acc.
Q08050
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.

Upstream / Downstream

pathwayImage

Explore pathways related to this product.

To Purchase # 54236S

View sizes
Product # Size Price
54236S
100 µl (10 western blots) $ 255.0