World Leading Company in

Transcription Factors Research

Active Motifs TransAM kits are:

ELISA-based assays for the detection of transcription factor activation in mammalian tissue and cell extracts.

TransAM
sensitive, quantitative ELISAs for transcription factor activation
Transcription factors are DNA-binding proteins that tightly regulate gene expression. They consist of two distinct domains - one that displays high affinity for a specific DNA sequence and one that confers transcriptional activity. Transcription factors are activated by phosphorylation of specific residues or by processing bound inhibitory proteins. Understanding and quantifying transcription factors is essential for the study of cell functions in relation to differentiation, brain activity, immune response, inflammation and various disease states. 

The TransAM Advantage
Historically, transcription factor studies have been conducted using gelshifts, 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 TransAM?

• 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 

• 96-stripwell format enables both high and low throughput 

TransAM Kits in two different formats - original and flexi
Active Motif offers two different formats of the TransAM Kits. The original TransAM format is perfect for assaying transcription factor binding to the consensus-binding site. These original 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 or chemiluminescent readout (See Figure 1). 

In addition, Active Motif also offers a new TransAM Flexi Kit that is perfect for those researchers interested in studying alternative binding sites for their transcription factor. In TransAM Flexi, the researcher synthesizes a biotinylated oligo or PCR product, which contains the transcription factor-binding site of choice. A stimulated nuclear extract is mixed with this biotinylated oligo and then transferred to the wells of a 96-well streptavidin coated plate, where the biotinylated oligo containing the activated transcription factor is captured. Next, a primary antibody specific for an epitope on the bound and active form of the transcription factor is added followed by subsequent incubation with secondary antibody and Developing Solution to provide an easily quantified, sensitive colorimetric readout (See Figure 2). 


Figure 1: Flowchart of TransAM process 

Figure 2: Flowchart of TransAM Flexi process 

* Technology covered under AAT-filed patents and licensed to Active Motif. Use of TransAM in NFkB-related drug discovery may be covered under U.S. Patent No. 6,150,090 and require a license from Ariad Pharmaceuticals (Cambridge, MA, USA).