Upstream / Downstream

pathwayImage

Explore pathways related to this product.

Friends and Family

25% Off

the purchase of 3 or more products

Shop

Questions?

Find answers on our FAQs page.

ANSWERS  

PhosphoSitePlus® Resource

  • Additional protein information
  • Analytical tools

LEARN MORE

REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Mouse IgG1
Image
Image
Image
Image

Flow Cytometry

Flow cytometric analysis of DLD-1 cells, untreated (blue) or UV-treated (100 mJ/cm2, 2 hr recovery; green) using Phospho-p53 (Ser15) (16G8) Mouse mAb (Alexa Fluor® 488 Conjugate) (solid lines) or concentration-matched Mouse (MOPC-21) mAb IgG1 Isotype Control (Alexa Fluor® 488 Conjugate) #4878 (dashed lines).

Learn more about how we get our images
Image
Image
Page

Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

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

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

  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 antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. 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: 407

Product Usage Information

Application Dilutions
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.

Specificity / Sensitivity

Phospho-p53 (Ser15) (16G8) Mouse mAb (Alexa Fluor® 488) detects endogenous levels of p53 only when phosphorylated at Ser15. The antibody does not cross-react with p53 phosphorylated at other sites.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser15 of human p53. The antibody was conjugated to Alexa Fluor® 488 under optimal conditions with an F/P ratio of 2-6.

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 human cells.


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

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

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

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

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

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

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

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


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
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.

9235
Phospho-p53 (Ser15) (16G8) Mouse mAb (Alexa Fluor® 488 Conjugate)