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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous 45 Rabbit IgG

Western Blotting

Western blot analysis of extracts from COS-7 cells, mock transfected (-) or transfected with human FoxP3 (hFoxP3; +), and human thymus using FoxP3 (D8O6C) Rabbit mAb.

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Flow Cytometry

Flow cytometric analysis of human peripheral blood mononuclear cells gated on CD4+ lymphocytes, showing FoxP3 expression in CD25+ cells using FoxP3 (D6O8C) Rabbit mAb (left), and corresponding absence of signal in CD25+ cells using concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, (right). Anti-rabbit IgG (H+L), (F(ab')2) Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody. Refer to the product specific protocol link indicated on the datasheet.

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Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.

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. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Prestained Protein Marker, Broad Range (Premixed Format): (#7720).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  16. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#7720, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product datasheet) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised November 2013

protocol id: 10

Western Blot Reprobing Protocol

Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. Wash Buffer: Tris Buffered Saline with Tween® 20 (TBST-10X) (#9997)
  2. Stripping Buffer: To prepare 100 ml, mix 0.76 g Tris base, 2 g SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H2O. Adjust pH to 6.8 with HCl.

B. Protocol

  1. After film exposure, wash membrane four times for 5 min each in TBST. Best results are obtained if the membrane is not allowed to dry.
  2. Incubate membrane for 30 min at 50°C in stripping buffer (with slight agitation).
  3. Wash membrane six times for 5 min each in TBST.
  4. (Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.
  5. Wash membrane again four times for 5 min each in TBST.
  6. The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

posted June 2005

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Flow Cytometry

A. Solutions and Reagents

NOTE: Prepare solutions with RODI (reverse osmosis deionized) 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. Formaldehyde (methanol free).
  3. 2% Formaldehyde (stock formaldehyde diluted in PBS; make fresh on day of experiment).
  4. Triton™ X-100.
  5. Ficoll-Paque™.
  6. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) (#9988) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: Surface staining of CD4 and CD25 antibodies should be performed on live cells prior to fixation/permeabilization, as per manufacturer’s requirements.

  1. Isolate PBMCs from whole blood by Ficoll density centrifugation as per manufacturer’s instructions.
  2. Wash 2x by centrifugation using Incubation Buffer.
  3. Resuspend cells in Incubation Buffer and aliquot at a concentration of 1 x 106 cells/100 µl/sample tube.
  4. Add CD4 and CD25 antibodies to assay tubes as per manufacturer’s recommended volume or concentration and incubate for 30 min. on ice.
  5. Add 2 ml of Incubation Buffer and wash by centrifugation.
  6. Aspirate supernatant and resuspend cells in 500 µl of 2% formaldehyde.
  7. Fix for 15 min at room temperature.
  8. Wash 2X by centrifugation in Incubation Buffer.

C. Permeabilization

  1. Resuspend cells in 1 ml of 0.1% Triton™ X-100 (v/v in PBS).
  2. Let stand for 30 min at room temperature.
  3. Wash 2X by centrifugation in Incubation Buffer.

D. Immunostaining

  1. Resuspend cell pellets in 100 µl of FoxP3 (D6O8C) XP® Rabbit mAb #12632 or FoxP3 (D6O8R) XP® Rabbit mAb #12653 working solution (stock antibody diluted 1:200 in Incubation Buffer).
  2. Incubate for 1 hr at room temperature.
  3. Wash 2X by centrifugation in Incubation Buffer.
  4. Resuspend cells in fluorochrome-conjugated secondary antibody, diluted in Incubation Buffer at the recommended dilution.
  5. Incubate for 30 min at room temperature.
  6. Wash 2X by centrifugation in Incubation Buffer.
  7. Resuspend cells in 500 µl of Incubation Buffer and analyze on flow cytometer.
  8. FoxP3 can be plotted against CD25 on a bivariate scattergram gated on CD4+ T lymphocytes.

posted July 2013

revised October 2013

protocol id: 63

Product Usage Information

Application Dilutions
Western Blotting 1:1000
Flow Cytometry 1:200

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.

Specificity / Sensitivity

FoxP3 (D6O8C) Rabbit mAb recognizes endogenous levels of total FoxP3 protein.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro293 of human FoxP3 protein.

Forkhead box (Fox) proteins are a family of evolutionarily conserved transcription factors containing a sequence known as Forkhead box or winged helix DNA binding domain (1). The human genome contains 43 Fox proteins that are divided into subfamilies. The FoxP subfamily has four members, FoxP1 - FoxP4, which are broadly expressed and play important roles in organ development, immune response and cancer pathogenesis (2-4). The FoxP subfamily has several characteristics that are atypical among Fox proteins: their Forkhead domain is located at the carboxy-terminal region and they contain motifs that promote homo- and heterodimerization. FoxP proteins usually function as transcriptional repressors (4,5).

FoxP3 is crucial for the development of T cells with regulatory properties (Treg) (6). Mutations in FoxP3 are associated with immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (IPEX) (7), while overexpression in mice causes severe immunodeficiency (8). Research studies have shown that FoxP3 functions as a tumor suppressor in several types of cancer (9-11).


1.  Myatt, S.S. and Lam, E.W. (2007) Nat Rev Cancer 7, 847-59.

2.  Shu, W. et al. (2001) J Biol Chem 276, 27488-97.

3.  Lu, M.M. et al. (2002) Gene Expr Patterns 2, 223-8.

4.  Koon, H.B. et al. (2007) Expert Opin Ther Targets 11, 955-65.

5.  Li, S. et al. (2004) Mol Cell Biol 24, 809-22.

6.  Ochs, H.D. et al. (2007) Immunol Res 38, 112-21.

7.  Bennett, C.L. et al. (2001) Nat Genet 27, 20-1.

8.  Kasprowicz, D.J. et al. (2003) J Immunol 171, 1216-23.

9.  Zuo, T. et al. (2007) Cell 129, 1275-86.

10.  Zuo, T. et al. (2007) J Clin Invest 117, 3765-73.

11.  Wang, L. et al. (2009) Cancer Cell 16, 336-46.

12.  Ochs, H.D. et al. (2007) Immunol Res 38, 112-21.

13.  Bennett, C.L. et al. (2001) Nat Genet 27, 20-1.

14.  Kasprowicz, D.J. et al. (2003) J Immunol 171, 1216-23.

15.  Zuo, T. et al. (2007) Cell 129, 1275-86.

16.  Zuo, T. et al. (2007) J Clin Invest 117, 3765-73.

17.  Wang, L. et al. (2009) Cancer Cell 16, 336-46.


Entrez-Gene Id 50943
Swiss-Prot Acc. Q9BZS1


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor® is a registered trademark of Life Technologies Corporation.