TransAM® p53 Transcription Factor ELISA Kits
TransAM p53 Kits provide everything needed to study activated p53 tumor suppressor, including a positive control extract. The kit can be used with human extracts. Recombinant p53 protein is available separately to generate an optional protein standard curve in the TransAM p53 Transcription Factor ELISA kits. See the p53 Info tab below for kit data and more information; the kit manual can be downloaded under the Documents tab.
|TransAM® p53 Manual|
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|Tools for Disease Research|
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|MSDS: Sulphuric Acid|
p53 Transcription Factor Info
p53, also known as tumor protein p53 (TP53), is the most important tumor suppressor in the genome. It is responsive to numerous genotoxic stresses, which activates its transcription factor activity to control the expression of genes that regulate cell growth control, DNA repair, cell-cycle arrest, apoptosis, angiogenesis, redox regulation, metastasis, nitric oxide production and protein degradation. Mutant p53 that has lost its DNA-binding function interferes with the activity of native p53 and leads to oncogenic transformation. Mutational inactivation of the p53 gene product is one of the most common genetic events that occur in human cancers. Alternatively, transformation may be caused by overexpression of Mdm2/Hdm2, a ubiquitin ligase specific for p53, which causes its destabilization.
Figure 1: Monitoring p53 activation with the TransAM p53 Kit.
The TransAM® transcription factor ELISA advantage
Historically, transcription factor studies have been conducted using gelshift, Western blot and reporter plasmid transfections, which are time-consuming, do not allow for high-throughput and provide only semi-quantitative results. TransAM assays are up to 100 times more sensitive than gelshift techniques, and can be completed in less than 5 hours. Because TransAM is an ELISA-based assay*, there is no radioactivity, and the high-throughput stripwell format enables simultaneous screening of 1-96 samples. Inconsistencies due to variable reporter plasmid transfections are eliminated, along with the need to construct stable cell lines.
Why use TransAM® transcription factor ELISAs?
- Up to 100-fold more sensitive than gelshift assays
- Eliminates the use of radioactivity and the need to run gels
- Results in less than five hours
- Colorimetric readout enables easy, quantitative analysis with spectrophotometry at 450 nm
- 96-stripwell format enables both high and low throughput
How TransAM® transcription factor ELISAs work
The TransAM format is perfect for assaying transcription factor binding to a consensus-binding site. TransAM Kits contain a 96-stripwell plate to which the consensus-binding site oligo has been immobilized. Activated nuclear extract is added to each well and the transcription factor of interest binds specifically to this bound oligonucleotide. A primary antibody specific for an epitope on the bound and active form of the transcription factor is then added followed by subsequent incubation with secondary antibody and Developing Solution to provide an easily quantified, sensitive colorimetric readout (Figure 1).
Figure 1: Flow chart of the TransAM process.
Contents & Storage
One or five p53 96-well assay plate(s) with plate sealer(s), p53 primary antibody, HRP-conjugated secondary antibody, p53 wild-type and mutated oligonucleotides, MCF-7 nuclear extract (positive control), Dithiothreitol, Protease Inhibitor Cocktail, poly[d(I-C)], Lysis, Binding, 10X Washing and 10X Antibody Binding Buffers, and Developing and Stop Solutions. Reagent storage conditions vary from room temperature to –80°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.
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