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Tools to analyze nuclear function,
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TransAM™ AML/Runx

 AML/Runx factors are essential for blood, skeletal and gastric development and are composed of heterodimeric α and β subunits. In mammals, the α subunits are encoded by three genes, Runx1, 2 and 3, while the β subunit (PEBP2β, CBFβ) is ubiquitously expressed. AML-1/Runx1 is essential for definitive hematopoiesis and is one of the most frequently mutated genes in human leukemia development, while AML-3/Runx2 controls osteoblast differentiation and the development of hypertrophic cartilage. Mutations of AML-3 lead to the rare bone disease cleidocranial dysplasia (CCD) and are associated with human leukemia.

 

 
Name Format Cat No. 価格 (税抜)  
TransAM® AML-1/Runx1 1 x 96 rxns 47396 ¥115,000 Buy Now
5 x 96 rxns 47896 ¥483,000 Buy Now
TransAM® AML-3/Runx2 1 x 96 rxns 44496 ¥115,000 Buy Now
5 x 96 rxns 44996 ¥483,000 Buy Now

AML/Runx Transcription Factor Info

AML/Runx factors are essential for blood, skeletal and gastric development and are composed of heterodimeric α and β subunits. In mammals, the α subunits are encoded by three genes, Runx1, 2 and 3, while the β subunit (PEBP2β, CBFβ) is ubiquitously expressed. AML-1/Runx1 is essential for definitive hematopoiesis and is one of the most frequently mutated genes in human leukemia development, while AML-3/Runx2 controls osteoblast differentiation and the development of hypertrophic cartilage. Mutations of AML-3 lead to the rare bone disease cleidocranial dysplasia (CCD) and are associated with human leukemia.

 
The TransAM AML-1/Runx1 Kit was used to test increasing amounts of nuclear extract from unstimulated HeLa and HL-60 cells for activated AML-1/Runx1
 
Figure 1: Monitoring AML-1/Runx1 activation with the TransAM AML-1/Runx1 Kit.

Increasing amounts of nuclear extract from unstimulated HeLa and HL-60 cells are tested for AML-1/Runx1 activation using the TransAM AML-1/Runx1 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 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. 

TransAM AML-1/Runx1: Catalog Number 44496
TransAM AML-1/Runx1: Catalog Number 44996
TransAM AML-1/Runx1: Catalog Number 47396
TransAM AML-1/Runx1: Catalog Number 47896