Upstream / Downstream

pathwayImage

Explore pathways related to this product.

Antibody Guarantee

CST Antibody Performance Guarantee

LEARN MORE  

To Purchase # 5429S

5429S 100 µl (50 tests) $299.00
$0.00

CUSTOM FORMULATION

Questions?

Find answers on our FAQs page.

ANSWERS  

PhosphoSitePlus® Resource

  • Additional protein information
  • Analytical tools

LEARN MORE

PRINT

p53 (7F5) Rabbit mAb (Alexa Fluor® 488 Conjugate) #5429

REACTIVITY SENSITIVITY MW (kDa) Isotype
H Mk Endogenous Rabbit IgG
Image
‹ 1 2 3 ›
Image
‹ 1 2 3 ›
Image
‹ 1 2 3 ›
IF-IC
enlarge

Immunofluorescence (Immunocytochemistry)

Confocal immunofluorescent analysis of HT-29 cells (positive) (left) and THP-1 cells (negative) (right) using p53 (7F5) Rabbit mAb (Alexa Fluor® 488 Conjugate) (green). Actin filaments have been labeled with DY-554 phalloidin (red).

Learn more about how we get our images
Image
‹ 1 2 3 ›
Flow Cytometry
enlarge

Flow Cytometry

Flow cytometric analysis of K562 (blue) and HT-29 cells (green) using p53 (7F5) Rabbit mAb (Alexa Fluor® 488 Conjugate).

Learn more about how we get our images
Image
‹ 1 2 3 ›
Image
‹ 1 2 3 ›
expand collapse

Protocol

Page
‹ 1 2 3 ›
PRINT

Immunofluorescence (Immunocytochemistry)

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 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
  5. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.

    NOTE: Formaldehyde is toxic, use only in a fume hood.

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  7. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised November 2013

Protocol Id: 182
Page
‹ 1 2 3 ›
PRINT

Flow Cytometry General Protocol

If using whole blood, please follow the Flow Cytometry Whole Blood Protocol.

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

  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 10 min at 37°C.
  4. Chill tubes on ice for 1 min.
  5. For extracellular staining with antibodies that do not require permeabilization, proceed to immunostaining (Section D) or store cells in PBS with 0.1% sodium azide at 4°C; for intracellular staining, proceed to permeabilization (Section C).

C. Permeabilization

NOTE: This step is critical for many CST antibodies.

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol. Alternatively, remove fix prior to permeabilization by centrifugation and resuspend in 90% methanol as described above.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

  1. Aliquot 0.5–1 x 106 cells into each assay tube (by volume).
  2. Add 2–3 ml incubation buffer to each tube and wash by centrifugation. Repeat.
  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 2–3 ml incubation buffer.
  6. Resuspend cells in 0.5 ml 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 30 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised September 2013

Flow Cytometry Whole Blood Protocol

If using cell lines, please follow the Flow Cytometry General Protocol.

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. Triton™ X-100: To prepare 50 ml of 0.1% Triton™ X-100 add 50 μl Triton™ X-100 to 50 ml 1 X PBS and mix well.
  4. 50% methanol.
  5. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Preparation of Whole Blood (fixation, lysis, and permeabilization) for Immunostaining

  1. Aliquot 100 μl fresh whole blood per assay tube.
  2. OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc.
  3. Add 65 μl of 10% formaldehyde to each tube.
  4. Vortex briefly and let stand for 15 min at room temperature.
  5. Add 1 ml of 0.1% Triton™ X-100 to each tube.
  6. Vortex and let stand for 30 min at room temperature.
  7. Add 1 ml incubation buffer.
  8. Pellet cells by centrifugation and aspirate supernatant.
  9. Repeat steps 7 and 8.
  10. Resuspend cells in ice-cold 50% methanol in PBS (store methanol solution at -20°C until use).
  11. Incubate at least 10 min on ice.
  12. Proceed with staining or store cells at -20°C in 50% methanol.

C. Staining Using Conjugated Primary Antibodies

NOTE: Account for isotype-matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies.

  1. Add 2–3 ml incubation buffer to each tube and rinse by centrifugation. Repeat.
  2. Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer.
  3. Incubate for 1 hr at room temperature.
  4. Wash by centrifugation in 2–3 ml incubation buffer.
  5. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

Reference: Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV (2005) Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67(1), 4–17.

posted November 2008

revised September 2013

Protocol Id: 407
expand collapse

Product Usage Information

Application Dilutions
Immunofluorescence (Immunocytochemistry) 1:50
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.
expand collapse

Specificity / Sensitivity

p53 (7F5) Rabbit mAb (Alexa Fluor® 488 Conjugate) detects endogenous levels of total p53 protein.


Species Reactivity: Human, Monkey
expand collapse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a MBP-p53 fusion protein.

expand collapse

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometry and immunofluorescent analysis in monkey cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated p53 (7F5) Rabbit mAb #2527.


expand collapse

Background

The p53 tumor suppressor protein plays a major role in cellular response to DNA damage and other genomic aberrations. Activation of p53 can lead to either cell cycle arrest and DNA repair or apoptosis (1). p53 is phosphorylated at multiple sites in vivo and by several different protein kinases in vitro (2,3). DNA damage induces phosphorylation of p53 at Ser15 and Ser20 and leads to a reduced interaction between p53 and its negative regulator, the oncoprotein MDM2 (4). MDM2 inhibits p53 accumulation by targeting it for ubiquitination and proteasomal degradation (5,6). p53 can be phosphorylated by ATM, ATR, and DNA-PK at Ser15 and Ser37. Phosphorylation impairs the ability of MDM2 to bind p53, promoting both the accumulation and activation of p53 in response to DNA damage (4,7). Chk2 and Chk1 can phosphorylate p53 at Ser20, enhancing its tetramerization, stability, and activity (8,9). p53 is phosphorylated at Ser392 in vivo (10,11) and by CAK in vitro (11). Phosphorylation of p53 at Ser392 is increased in human tumors (12) and has been reported to influence the growth suppressor function, DNA binding, and transcriptional activation of p53 (10,13,14). p53 is phosphorylated at Ser6 and Ser9 by CK1δ and CK1ε both in vitro and in vivo (13,15). Phosphorylation of p53 at Ser46 regulates the ability of p53 to induce apoptosis (16). Acetylation of p53 is mediated by p300 and CBP acetyltransferases. Inhibition of deacetylation suppressing MDM2 from recruiting HDAC1 complex by p19 (ARF) stabilizes p53. Acetylation appears to play a positive role in the accumulation of p53 protein in stress response (17). Following DNA damage, human p53 becomes acetylated at Lys382 (Lys379 in mouse) in vivo to enhance p53-DNA binding (18). Deacetylation of p53 occurs through interaction with the SIRT1 protein, a deacetylase that may be involved in cellular aging and the DNA damage response (19).


1.  Chehab, N.H. et al. (1999) Proc Natl Acad Sci U S A 96, 13777-82.

2.  Levine, A.J. (1997) Cell 88, 323-31.

3.  Meek, D.W. (1994) Semin Cancer Biol 5, 203-10.

4.  Milczarek, G.J. et al. (1997) Life Sci 60, 1-11.

5.  Shieh, S.Y. et al. (1997) Cell 91, 325-34.

6.  Tibbetts, R.S. et al. (1999) Genes Dev 13, 152-7.

7.  Honda, R. et al. (1997) FEBS Lett 420, 25-7.

8.  Shieh, S.Y. et al. (1999) EMBO J 18, 1815-23.

9.  Hirao, A. et al. (2000) Science 287, 1824-7.

10.  Hao, M. et al. (1996) J Biol Chem 271, 29380-5.

11.  Lu, H. et al. (1997) Mol Cell Biol 17, 5923-34.

12.  Ullrich, S.J. et al. (1993) Proc Natl Acad Sci U S A 90, 5954-8.

13.  Kohn, K.W. (1999) Mol Biol Cell 10, 2703-34.

14.  Lohrum, M. and Scheidtmann, K.H. (1996) Oncogene 13, 2527-39.

15.  Knippschild, U. et al. (1997) Oncogene 15, 1727-36.

16.  Oda, K. et al. (2000) Cell 102, 849-62.

17.  Ito, A. et al. (2001) EMBO J 20, 1331-40.

18.  Sakaguchi, K. et al. (1998) Genes Dev 12, 2831-41.

19.  Solomon, J.M. et al. (2006) Mol Cell Biol 26, 28-38.


Entrez-Gene Id 7157
Swiss-Prot Acc. P04637
expand collapse

Data Sheets & Documentation

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
The Alexa Fluor dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.
U.S. Patent No. 5,675,063.

5429
p53 (7F5) Rabbit mAb (Alexa Fluor® 488 Conjugate)
ABOUT
LEGAL
SUPPORT
LOCATIONS

    Cell Signaling Technology

    3 Trask Lane

    Danvers, MA 01923

    Global Offices
JOBS
SIGN UP FOR NEWS
VISIT US AT
© 2016 Cell Signaling Technology, Inc. All Rights Reserved. Email info@cellsignal.com