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.
- 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
- 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
- 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
<lIκBenchtop rocker or shaker
- Microplate reader capable of measuring absorbance at 450 nm
- Prepare all reagents and samples as instructed in the manual.
- Add 100 µl of sample or positive control to each well.
- Incubate 2 h at RT or O/N at 4 °C.
- Add 100 µl of prepared primary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of prepared HRP-secondary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of TMB One-Step Substrate Reagent to each well.
- Incubate 30 min at RT.
- Add 50 µl of Stop Solution to each well.
- Read at 450 nm immediately.
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 with the specific competitor or non-specific competitor. The result shows specific binding of p53 to the p53 DNA binding site.
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.