Human p53 Transcription Factor Activity Assay

RayBio® Human p53 Transcription Factor Activity Assay. This assay uses a dsDNA coated plate with canonical p53 binding sequences to semi-quantitatively detect active p53 in lysates or nuclear extracts. Dry ice shipment (additional fee).

$723.60  
TFEH-p53-2
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Antigen Information

Accession Number:
  • P04637
Gene ID:
  • 7157
Gene Symbols:
  • P53
  • TP53
Protein Name & Synonyms:
Cellular tumor antigen p53 (Antigen NY-CO-13) (Phosphoprotein p53) (Tumor suppressor p53)
Target Species:
  • Human

Assay Format

Pathway:
  • Akt Signaling
  • AMPK Signaling
  • HER/ErbB Signaling
  • HIF-1 alpha Signaling
  • MAPK Signaling
  • mTOR Signaling
  • p53 Signaling
  • PI3K-AKT Signaling
Specificity:
The olionucleotide/antibody pair provided in this kit recognizes human p53 in whole lysates and nuclear extracts.
Number of Targets Detected:
1
Species Detected:
  • Human
Compatible Sample Types:
  • Cell Lysates
  • Nuclear Extracts
Design Principle:
  • Sandwich-based
Method of Detection:
Colorimetric
Quantitative/Semi-Quantitative:
  • Semi-Quantitative
Solid Support:
96-well Microplate

Product Specifications

Size:
1, 2, or 5 x 96-Well Strip Microplate Kit

Introduction

p53 is a master regulating transcription factor of cell fate that activates genes responsible for a cell-cycle checkpoint or apoptosis after exposure to ionizing radiation, UV light, or other DNA-damaging agents. To date over 125 protein-coding genes and noncoding RNAs have been shown to be a direct transcriptional target of p53. The p53 protein has features commonly associated with transcriptional regulators including an amino-terminal transactivation domain consisting of two transactivation subdomain TAD-I and TAD-II, a proline-rich region, a conserved core DNA-binding domain (DBD), a linker region, a tetramerization domain, and an unstructured basic domain in the carboxy-terminus. In normal cells where p53 is found at very low levels, p53 is present in a complex with MDM2, an E3 ubiquitin ligase, which targets p53 for degradation through the ubiquitin pathway. In response to stimuli, this inhibition is relieved and p53 is activated through a number of mechanisms which include phosphorylation, dephosphorylation by protein serine/threonine phoshatase-1, acetylation by the transcriptional coactivator p300/CBP, and induced conformational changes mediated by the prolyl isomerase Pin1. An increase in active p53 protein levels leads to multiple outcomes such as cell cycle arrest, apoptosis, senescence, autophagy, and others. Contrarily, the loss or inactivation of p53 due to damage or mutation, results in the loss of cell-cycle arrest or apoptosis after DNA damage or physiologic stresses. This loss, seen in many human cancers, is expected to lead to increased genetic instability, increased accumulation of mutations, and ultimately oncogenesis.

Product Features

  • Specific transcription factor-DNA binding assay
  • Perfect alternative to EMSA
  • Easy to perform in an ELISA format
  • Non-radioactive assay
  • High throughput (96-well plate format)
  • Assay can be completed within 5 hours

Application Notes

Kit Components
  • 96-well Strip Microplate pre-coated with DNA probes
  • DNA Binding Buffer
  • Positive Control Sample
  • Specific Competitor DNA probe
  • Non-specific Competitor DNA probe
  • Assay Reagent
  • DTT
  • Wash Buffer
  • Primary Antibody
  • HRP-conjugated Secondary Antibody
  • Antibody Diluent Buffer
  • TMB One-Step Substrate Reagent
  • Stop Solution
Other Materials Required
  • Distilled or deionized water
  • 100 ml and 1 liter graduated cylinders
  • Tubes to prepare sample dilutions
  • Absorbent paper
  • Precision pipettes to deliver 2 µl to 1 ml volumes
  • Adjustable 1-25 ml pipettes for reagent preparation
  • Microplate reader capable of measuring absorbance at 450 nm
Protocol Outline
  1. Prepare all reagents and samples as instructed in the manual.
  2. Add 100 µl of sample or positive control to each well.
  3. Incubate 2 h at RT or O/N at 4 °C.
  4. Add 100 µl of prepared primary antibody to each well.
  5. Incubate 1 h at RT.
  6. Add 100 µl of prepared HRP-secondary antibody to each well.
  7. Incubate 1 h at RT.
  8. Add 100 µl of TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 µl of Stop Solution to each well.
  11. Read at 450 nm immediately.

Typical Data

Figure 1
Transcription factor assay of p53 from nuclear extracts of HeLa cells or HeLa cells treated with NiCl2. After stimulation activated p53 binds with its corresponding DNA. A. Western-blot result of p53 from cytoplasm and nuclear fractions. B. Transcription factor assay of p53 from nuclear fractions with RayBio® p53 TF-Activity Assay Kit .

Transcription factor assay of p53 from nuclear extracts of HeLa cells or HeLa cells treated with NiCl2. After stimulation activated p53 binds with its corresponding DNA. A. Western-blot result of p53 from cytoplasm and nuclear fractions. B. Transcription factor assay of p53 from nuclear fractions with RayBio p53 TF-Activity Assay Kit

Figure 2
Transcription factor assay of p53 from nuclear extracts of HeLa cells or HeLa cells treated with NiCl2 with the specific competitor or non-specific competitor. The result shows specific binding of p53 to the p53 DNA binding site.

Transcription factor assay of p53 from nuclear extracts of HeLa cells or HeLa cells treated with NiCl2 with the specific competitor or non-specific competitor. The result shows specific binding of p53 to the p53 DNA binding site.

Storage/Stability

Upon receipt, the positive control should be removed and stored at -20° or -80°C. The remainder of the kit can be stored for up to 6 months at 2-8°C from the date of shipment. Opened Microplate Wells or reagents may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge.
Note: The kit can be used within one year if the whole kit is stored at -20°C upon receipt. Avoid repeated freeze-thaw cycles.

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Usage

This product is furnished for LABORATORY RESEARCH USE ONLY.
Not for diagnostic or therapeutic use.

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