dna-binding ELISAs for activated T-bet transcription factor
TransAM® Kits are DNA-binding ELISAs that facilitate the study of transcription factor activation in mammalian tissue and cell extracts. Assays are available for over 40 different targets (see the list at right). Each kit includes a 96-stripwell plate in which multiple copies of a specific double-stranded oligonucleotide have been immobilized. When nuclear or whole-cell extract is added, activated transcription factor of interest binds the oligonucleotide at its consensus binding site and is quantified using the included antibody, which is specific for the bound, active form of the transcription factor being studied. For complete details, click the TransAM® Method tab below.
TransAM® T-bet Transcription Factor ELISA Kits
TransAM T-bet Kits provide everything needed to study T-box transcription factor expressed in T-cells (T-bet or TBX21), including a positive control extract. The kit can be used with human extracts. See the T-bet Info tab below for kit data and more information; the kit manual can be downloaded under the Documents tab.
|TransAM® T-bet||1 x 96 rxns||51396||$685||Buy Now|
|5 x 96 rxns||51896||$2,795||Buy Now|
|TransAM® T-bet Manual|
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T-bet Transcription Factor Info
T-box transcription factor expressed in T cells (T-bet), also known as T-box 21 (TBX21) is a transcription factor essential to T helper 1 (Th1) cell generation and effector function. Recently, the expression of T-bet has also been described in B, natural killer (NK), CD8+ T and dendritic cells. T-bet regulates expression of numerous immune system-associated genes, including cytokines, cytokine receptors, chemokines, chemokine receptors and cytotoxicity.
Figure 1: Monitoring T-bet activation with the TransAM T-bet 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 96-well plate(s) with plate sealer(s), primary antibody, HRP-conjugated secondary antibody, wild-type and mutated oligonucleotides, positive control cell extract, DTT, Herring Sperm DNA, Protease Inhibitor Cocktail, 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.