The total p53 kit detects cellular p53 and can be used as a normalization assay for the phospho-p53 kit. It is optimal for monitoring cellular response to DNA damage.
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  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • No-wash No-wash
The total p53 kit detects cellular p53 and can be used as a normalization assay for the phospho-p53 kit. It is optimal for monitoring cellular response to DNA damage.
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Overview

The Total p53 cellular assay kit serves as a normalization assay with our Phospho-p53 kit. 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.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total-p53 assay principle

The Total-p53 assay quantifies the expression level of p53 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-p53 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of p53 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
Total p53 assay principle

Total-p53 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 Total p53 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total p53 2-plate assay protocol

Total-p53 1-plate assay protocol

Detection of total p53 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.
Total p53 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.
HTRF assay vs WB using phospho & total P53 assays on human MCF-7 cells

Validation of Total-p53 assay 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.
Validation on Neocarcinostatin treated MCF-7 breast cancer cells

Validation of Total-p53 assay 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.
Validation on Etoposide treated breast cancer MCF-7 cells

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.
Function and regulation of p53
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Product Insert P53 total Kit / 64T53PEG-64T53PEH

64T53PEG-64T53PEH - Product Insert

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

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