Product Pathways - PathScan ELISA
PathScan® Total p53 Sandwich ELISA Kit #7370
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|Kit Includes||Volume||Solution Color|
|p53 Rabbit mAb Coated Microwells|
|p53 Detection Ab||11 ml||Green|
|Anti-mouse IgG, HRP-linked Antibody||11 ml||Red|
|TMB Substrate #7004||11 ml||Colorless|
|STOP Solution #7002||11 ml||Colorless|
|Sealing Tape||2 sheets|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) #9803||15 ml||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, which is stored at –20°C (packaged separately).
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
CST's PathScan® Total p53 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total p53 protein. A p53 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 p53 Mouse mAb is added to detect the captured 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 total p53 protein.Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Total p53 Sandwich ELISA Kit detects endogenous levels of total p53 protein. Using PathScan® Phospho-p53 (Ser15) Sandwich ELISA Kit #7365, a significant induction of phospho-p53 in HT-29 cells treated with UV can be detected. However, the level of total p53 (phospho and non-phospho), detected by this Sandwich ELISA Kit #7370, remains unchanged (Figure 1). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
ELISA - Western correlation
Figure 1: Treatment of HT-29 cells with UV stimulates phosphorylation of p53 at Ser15, detected by PathScan® Phospho-p53 (Ser15) Sandwich ELISA kit, #7365, but does not affect the level of total p53 protein detected by PathScan® Total p53 Sandwich ELISA kit, #7370. OD450 readings are shown in the top figure, while the corresponding Western blot using Phospho-p53 (Ser15) Mouse mAb #9286 (right panel) or p53 Mouse mAb #2524 (left panel), is shown in the bottom figure.
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).
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- 7365 PathScan® Phospho-p53 (Ser15) Sandwich ELISA Kit
- 2524 p53 (1C12) Mouse mAb
- 9286 Phospho-p53 (Ser15) (16G8) Mouse mAb
- 7076 Anti-mouse IgG, HRP-linked Antibody
- 9803 Cell Lysis Buffer (10X)
- 7004 TMB Substrate
- 7002 STOP Solution
Rabbit Monoclonals Produced UsingTechnology from Epitomics, Inc. UnderU.S. patent No. 5,675,063.
For Research Use Only. Not For Use In Diagnostic Procedures.