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TransAM® c-Myc

dna-binding ELISA for activated c-Myc transcription factor

TransAM c-Myc Kits provide everything needed to study activated c-Myc, including a positive control extract. The c-Myc kit can be used with human and mouse extracts. TransAM c-Myc Kits contain an antibody specific for the active form of c-Myc when bound to its target DNA. Recombinant c-Myc protein is available separately to generate an optional protein standard curve. The Myc gene is a proto-oncogene that codes for a transcription factor that regulates cell growth, differentiation, glycolysis and apoptosis. c-Myc is a cellular homolog of the avian retroviral v-myc oncogene and belongs to the Myc family, which also includes N-myc and L-myc. Translocations and deregulation of c-Myc have been implicated in cancers such as hematopoietic tumors, leukemias and lymphomas.  Heterodimer formation of c-Myc with Max (Myc associated factor X) is required to bind DNA at the E-box motif CAC[GA]TG and activate transcription. c-Myc is activated by the MAP Kinase ERK1/2. Because of c-Myc's regulation by MAPK, we also offer the TransAM MAPK Family Kit for studying the MAPK regulated transcription factors ATF-2, c-Jun, c-Myc, MEF2 and STAT1α. The c-Myc Respone Element is available as a pre-cloned transfection ready luciferase reporter vector on the LightSwitch™ Reporter System page under Synthetic Response Element Collection.

The TransAM® Method tab below provides complete method details; the kit manual can be downloaded under the Documents tab, while a selection of papers that cite the use of TransAM c-Myc can be seen under the Publications tab.

 
Measurement of c-Myc activity 
Measurement of c-Myc activity.

Different amounts of nuclear extracts from untreated Jurkat and U-937 cells are tested for c-Myc activity using the TransAM c-Myc Kit.

 
Name Format Cat No. 価格 (税抜)  
TransAM® c-Myc 1 x 96 rxns 43396 ¥115,000 Buy Now
5 x 96 rxns 43896 ¥483,000 Buy Now

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 c-Myc 96-well plate(s) with plate sealer(s), primary antibody, HRP-conjugated secondary antibody, wild-type and mutated competitor oligonucleotides, positive control cell extract, DTT, 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 c-Myc: Catalog Number 43396
TransAM c-Myc: Catalog Number 43896