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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Mk Endogenous Rabbit IgG
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Immunofluorescence (Immunocytochemistry)

Confocal immunofluorescent analysis of HT-29 cells (positive) (left) and THP-1 cells (negative) (right) using p53 (7F5) Rabbit mAb (Alexa Fluor® 555 Conjugate) (red) and α-Tubulin (DM1A) Mouse mAb #3873 (green).

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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

Product Usage Information

Application Dilutions
Immunofluorescence 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.

Specificity / Sensitivity

p53 (7F5) Rabbit mAb (Alexa Fluor® 555 Conjugate) detects endogenous levels of total p53 protein.


Species Reactivity: Human, Monkey

Source / Purification

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

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 555 fluorescent dye and tested in-house for immunofluroescent analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated p53 (7F5) Rabbit mAb #2527.


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 NH 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, 203-210.

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

5.  Shieh, S.Y. et al. (1997) Cell 91, 325-334.

6.  Honda, R. et al. (1997) FEBS Lett. 420, 25-27.

7.  Tibbetts, R.S. et al. (1999) Genes Dev. 13, 152-157.

8.  Shieh, S.Y. et al. (1999) EMBO J. 18, 1815-1823.

9.  Hirao, A. et al. (2000) Science 287, 1824-1827.

10.  Hao, M. et al. (1996) J. Biol. Chem. 271, 29380-29385.

11.  Lu, H. et al. (1997) Mol. Cell. Biol. 17, 5923-5934.

12.  Ullrich, S.J. et al. (1993) Proc. Natl. Acad. Sci. USA 90, 5954-5958.

13.  Kohn, K.W. (1999) Mol. Biol. Cell 10, 2703-2734.

14.  Lohrum, M. and Scheidtmann, K.H. (1996) Oncogene 13, 2527-2539.

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

16.  Knippschild, U. et al. (1997) Oncogene 15, 1727-1736.

17.  Oda, K. et al. (2000) Cell 102, 849-862.

18.  Ito, A. et al. (2001) EMBO J. 20, 1331-1340.

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


Entrez-Gene Id 7157
Swiss-Prot Acc. P04637

Protein Specific References

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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.