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TransAM® AP-1

dna-binding ELISAs for c-Fos, FosB, Fra-1, c-Jun, JunB & JunD

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® AP-1 Transcription Factor ELISA Kits

TransAM AP-1 Kits provide everything needed to study activated AP-1, including a positive control extract. Kits are available with antibodies specific for the activated form of c-Fos, FosB, JunD or phosphorylated c-Jun. A family kit is also available with the ability to independently screen the members of the AP-1 family (c-Fos, FosB, Fra-1, c-Jun, JunB & JunD) simultaneously. The Fra-1 and JunB antibodies can be used with human extracts, while the c-Fos, FosB and JunD antibodies can be used with human, mouse and rat extracts. c-Jun phosphorylated at Ser73 and JunD phosphorylated at Ser100 can be detected in human and mouse extracts, as this site is conserved between c-Jun and JunD. Recombinant c-Fos and Recombinant c-Jun proteins are available separately to generate an optional protein standard curve in the TransAM AP-1 Transcription Factor ELISA kits. Click the AP-1 Info tab below for data and more information; kit manuals can be downloaded under the Documents tab.

 
Name Format Cat No. Price  
TransAM® AP-1 Family 2 x 96 rxns 44296 $1,125 Buy Now
TransAM® AP-1 c-Fos 1 x 96 rxns 44096 $665 Buy Now
5 x 96 rxns 44596 $2,795 Buy Now
TransAM® AP-1 c-Jun 1 x 96 rxns 46096 $665 Buy Now
5 x 96 rxns 46596 $2,795 Buy Now
TransAM® AP-1 FosB 1 x 96 rxns 45096 $665 Buy Now
5 x 96 rxns 45596 $2,795 Buy Now
TransAM® AP-1 JunD 1 x 96 rxns 43496 $665 Buy Now
5 x 96 rxns 43996 $2,795 Buy Now

AP-1 Transcription Factor Info

AP-1 proteins play a role in the expression of many genes involved in the regulation of cellular processes such as differentiation, proliferation and apoptosis. The transcription factor AP-1 is composed of a mixture of heterodimeric protein complexes derived from the Fos and Jun families, including c-Fos, FosB, Fra-1, c-Jun, JunB and JunD. AP-1 heterodimers bind to DNA on a serum response element with the 5´-TGA(C/G)TCA-3´ sequence. In addition to forming transcriptionally active homodimers with AP-1 members, Jun proteins can also bind to form transcriptionally active heterodimers with CREB/ATF members to bind the CRE element. AP-1 expression is induced by multiple stimuli, including the presence of serum, growth factors, phorbol esters and oncogenes. Phosphorylation of AP-1 family members by kinases is required for transactivation activity.

Variety of kits available

  • c-Fos
  • FosB
  • c-Jun
  • JunD
  • Family Kit (c-Fos, FosB, Fra-1, c-Jun, JunB and JunD)
 
TransAM AP1 family specificity 
 
Figure 1: AP-1 family profiling for DNA binding activation upon stimulation with TPA.

Nuclear extracts from K-562 cells stimulated with TPA were assayed for activity of AP-1 family members c-Jun, c-Fos, FosB, JunB, JunD and Fra-1. Each antibody was tested with 5 µg/well of nuclear extract in the absence or presence of wild-type or mutated consensus binding oligonucleotides using the TransAM AP-1 Family 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, two or five AP-1 96-well plate(s) with plate sealer(s), c-Fos, FosB, c-Jun, JunD or c-Fos, FosB, Fra-1, c-Jun, JunB and JunD primary antibody(ies), HRP-conjugated secondary antibody, AP-1 wild-type and mutated oligonucleotides, positive control cell extract, DTT, Protease Inhibitor Cocktail, Poly[d(I-C)], Lysis, Binding, 10X Washing and 10X Antibody Binding Buffer, 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.