Product Pathways - PathScan ELISA

PathScan® Phospho-p53 (Ser15) Sandwich ELISA Kit #7365

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Kit Includes	Volume	Solution Color
p53 (7365-15A5) Rabbit mAb Coated Microwells
Phospho-p53 (Ser15) Detection Ab	11 milliliters	Green
Anti-mouse IgG HRP-Linked Ab	11 milliliters	Red
TMB Substrate	11 milliliters	Colorless
STOP Solution	11 milliliters	Colorless
Sealing Tape	2 sheets
20X Wash Buffer	25 milliliters	Colorless
Sample Diluent	25 milliliters	Blue
Cell Lysis Buffer (10X) # 9803	15 milliliters	Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer (10X), which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human

Description

CST's PathScan® Phospho-p53 (Ser15) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Phospho-p53 (Ser15) protein. A p53 (7365-15A5) Rabbit mAb* has been coated onto the microwells. After incubation with cell lysates, Both nonphospho- and phospho-p53 proteins are captured by the coated antibody. Following extensive washing, a phospho-p53 (16G8) Mouse mAb #9286* is added to detect the captured phospho-p53 protein. HRP-linked anti-mouse antibody #7076* is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of phospho-p53 protein.* Antibodies in this kit are custom formulations specific to the kit.

Specificity / Sensitivity

CST's PathScan® Phospho-p53 (Ser15) Sandwich ELISA Kit detects endogenous levels of Phospho-p53 (Ser15) protein. Using this Sandwich ELISA Kit #7365, a significant induction of phospho-p53 (Ser15) in HT-29 cells treated with UV can be detected. However, the level of total p53 (phospho- and non-phospho), detected by PathScan® Total p53 Sandwich ELISA Kit #7370, remains unchanged (Figure 1).

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 (6,7). 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 (4,5). Chk2 and Chk1 can phosphorylate p53 at Ser20, enhancing its tetramerization, stability and activity (8,9). p53 is phosphorylated at Ser392 in vivo (11,12) and by CAK in vitro (12). Phosphorylation of p53 at Ser392 is increased in human tumors (14) and has been reported to influence the growth suppressor function, DNA binding and transcriptional activation of p53 (10,11,13). p53 is phosphorylated at Ser6 and Ser9 by CK1δ and CK1ε both in vitro and in vivo (10,15). 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, 323-331.
2. Meek, D.W. (1994) Semin. Cancer Biol. 5, 203-210.
3. Milczarek, G.J. et al. (1997) Life Sci. 60, 1-11.
4. Shieh, S.Y. et al. (1997) Cell 91, 325-334.
5. Tibbetts, R.S. et al. (1999) Genes Dev. 13, 152-157.
6. Chehab, N.H. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 13777-13782.
7. Honda, R. et al. (1997) FEBS Lett. 420, 25-27.
8. Shieh, S.Y. et al. (1999) EMBO J. 18, 1815-1823.
9. Hirao, A. et al. (2000) Science 287, 1824-1827.
10. Kohn, K.W. (1999) Mol. Biol. Cell 10, 2703-2734.
11. Hao, M. et al. (1996) J. Biol. Chem. 271, 29380-29385.
12. Lu, H. et al. (1997) Mol. Cell. Biol. 17, 5923-5934.
13. Lohrum, M. and Scheidtmann, K.H. (1996) Oncogene 13, 2527-2539.
14. Ulrich, S.J. et al. (1993) Proc. Natl. Acad. Sci. USA 90, 5954-5958.
15. Knippschild, U. et al. (1997) Oncogene 15, 1727-1736.
16. Oda, K. et al. (2000) Cell 102, 849-862.
17. Ito, A. et al. (2001) EMBO J. 20, 1331-1340.
18. Sakaguchi, K. et al. (1998) Genes Dev. 12, 2831-2841.
19. Solomon, J.M. et al. (2006) Mol. Cell. Biol. 26, 28-38.

Application References

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Companion Products

• 7370 PathScan® Total p53 Sandwich ELISA Kit
• 9286 Phospho-p53 (Ser15) (16G8) Mouse mAb
• 7076 Anti-mouse IgG, HRP-linked Antibody
• 9803 Cell Lysis Buffer (10X)
• 2524 p53 (1C12) Mouse mAb

Rabbit Monoclonals Produced UsingTechnology from Epitomics, Inc. UnderU.S. patent No. 5,675,063.

This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.