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HTRF Human Phospho-p53 (Ser15) Detection Kit, 500 Assay Points

The phospho-p53 (Ser15) kit enables the cell-based quantitative detection of phosphorylated p53 as a readout of the stress signaling pathway.

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption & disposal requirements under European REACH regulations (EC 1907/2006).
Part Number: 64P53PET
Unit Size: 96 Assay Points
Part Number: 64P53PEG
Unit Size: 500 Assay Points
Part Number: 64P53PEH
Unit Size: 10,000 Assay Points
  • This HTRF cell based assay conveniently and accurately quantifies phosphorylated p53 at Ser15. P53 has been described as the guardian of the genome because of its role in safeguarding genome integrity. P53 has many anticancer function mechanisms, influencing apoptosis, senescence, cell cycle regulation, growth arrest, genomic stability, angiogenesis inhibition, and metabolism changes, by regulating the expression of various target genes.

  • Assay Points
    500
    Assay Target Type
    Kit
    Assay Technology
    HTRF
    Brand
    HTRF
    Quantity
    1
    Therapeutic Area
    Oncology & Inflammation
    Unit Size
    500 Assay Points
  • Phospho-p53 (Ser15) assay principle

    The Phospho-p53 (Ser15) assay measures p53 when phosphorylated at Ser15. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-p53 (Ser15) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the proteins phosphorylation state under a no-wash assay format.

    phospho-p53-ser15-assay-principle

     

    Phospho-p53 (Ser15) 2-plate assay protocol

    The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Phospho-p53 (Ser15) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

    phospho-p53-ser15-2-plate-assay-protocol

     

    Phospho-p53 (Ser15) 1-plate assay protocol

    Detection of Phosphorylated p53 (Ser15) with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

    phospho-p53-ser15-1-plate-assay-protocol

     

  • HTRF assay vs WB using Phospho & total P53 assays on human MCF-7 cells

    Human MCF-7 cells were plated at 106 cells/ well in a 6 well plate, and incubated for 24h at 37°C, 5% CO2. After treatment for 5 min under UV (2.1J/cm²), the cells were lysed with 200 µL of lysis buffer for 30min at RT under gentle shaking. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-p53 (Ser15) detection reagents. Equal amounts of lysates were used for a side by side comparison of Western Blot and HTRF. Results show that the two HTRF p-53 assays are at least 4-fold more sensitive than the Western Blot.

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    Validation on Neocarcinostatin treated MCF-7 breast cancer cells

    Human MCF-7 cells were plated at 100,000 cells/ well in a 96 well plate, and incubated for 24h at 37°C, 5% CO2. After treatment for 45 min with increasing concentrations of Neocarcinostatin, the medium was removed and the cells were lysed with 50 µL of lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-p53 (Ser15) or total p53 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

    assay-validation-p53-phospho-s15-2

     

    Validation on Etoposide (apoptosis inductor) treated breast cancer MCF-7 cells

    Human MCF-7 cells were plated at 200,000 cells/ well in a 96 well plate, and incubated for 24h at 37°C, 5% CO2. After treatment for 3 h with increasing concentrations of Etoposide, the medium was then removed and the cells were lysed with 50 µL of lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-p53 (Ser15) or total p53 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

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  • Function and regulation of p53

    P53 becomes activated by a myriad of stressors including, but not limited to, DNA damage, oxidative stress, osmotic shock, ribonucleotide depletion, and deregulated oncogene expression. In response to such stresses, p53 undergoes extensive posttranslational modifications, including phosphorylation and acetylation. Phosphorylation of p53 mostly occurs in the N-terminal at Ser15 and leads to a reduced interaction between p53 and its negative regulator, the oncoprotein MDM2, which inhibits p53 accumulation by targeting it for ubiquitination and proteasomal degradation. Phosphorylation in this site impairs the ability of MDM2 to bind p53, promoting both the accumulation and activation of p53 in response to DNA damage.

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