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TransAM® MyoD

dna-binding ELISA for activated MyoD 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® MyoD Transcription Factor ELISA Kits

TransAM MyoD Kits provide everything needed to study activated Myogenic Differentiation 1 (MyoD), including a positive control extract. The MyoD Kit can be used with human, mouse and rat extracts. Click the MyoD Info tab below for data and more information; kit manuals can be downloaded under the Documents tab.

 
Name Format Cat No. Price  
TransAM® MyoD 1 x 96 rxns 47196 $665 Buy Now
5 x 96 rxns 47696 $2,795 Buy Now

MyoD Transcription Factor Info

Myogenic differentiation1 (MyoD), also known as myogenic factor 3 (Myf-3) is a transcription factor belonging to the myogenic factors family that contains a basic helix-loop-helix (bHLH) DNA-binding domain. The MyoD transcription factor is the central target in the signaling pathways that regulate muscle development. Although MyoD family members have been studied for decades in several muscle cell systems, the overall muscle differentiation program is still a strong area of focus to understand muscle decay.

 

 
 
Figure 1: Monitoring MyoD activity with the TransAM MyoD Kit:

Increasing amounts of nuclear extracts from confluent C2C12 cells (filled circles) and from NIH3T3 cells (open circles) are tested for MyoD activity using the TransAM MyoD 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).

Flow chart of the TransAM DNA binding transcription factor ELISA method for measurement of activated transcription factors
Figure 1: Flow chart of the TransAM process.

Activated transcription factor in the cell extract binds to the consensus-binding site on the oligo immobilized in the well. Incubation with the supplied primary and secondary antibodies specifically quantifies the amount of activated transcription factor.

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

Contents & Storage

One or five MyoD 96-well assay plate(s) with plate sealer(s), MyoD primary antibody, HRP-conjugated secondary antibody, wild-type and mutated oligonucleotides, C2C12 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.