TransAM® MAPK Family
dna-binding ELISA for activated ATF-2, c-Jun, c-Myc, MEF2 and STAT1 transcription factors
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® MAPK Family Transcription Factor ELISA Kits
TransAM MAPK Family Kits provide everything needed to study transcription factors activated by Mitogen-Activated Protein Kinase (MAPK) signal transduction pathways, including a positive control extract. The MAPK Family Kit contains multiple binding sites in each well and includes antibodies specific for the active forms of the transcription factors: phosphorylated ATF-2 (Thr71), phosphorylated c-Jun (Ser73), c-Myc, MEF2 and STAT1α, all of which are regulated by MAP kinase cascades and are key markers in cell signaling. In addition to the TransAM MAPK Family Kit, individual kits for studying ATF-2, c-Jun, c-Myc and MEF2 are also available. The phosphorylated ATF-2, MEF2 and STAT1α antibodies can be used with human extracts, while the phosphorylated c-Jun and c-Myc antibodies can be detected in human and mouse extracts. Recombinant c-Fos, Recombinant c-Jun and Recombinant c-Myc proteins are available separately to generate an optional protein standard curve in the TransAM Transcription Factor ELISA kits. Click the MAPK Info tab below for data and more information; kit manuals can be downloaded under the Documents tab.
|TransAM® MAPK Family Manual|
|Gene Regulation Products & Services Brochure|
|Tools for Disease Research|
|MSDS: Sodium Azide|
|MSDS: Sulphuric Acid|
The activation of Mitogen-Activated Protein Kinase (MAPK) signal transduction pathways is responsible for the phosphorylation of transcription factors by the terminal kinase in these cascades. MAPK pathways play major roles in converting mitogenic and stress stimuli into nuclear responses. At least three parallel MAPK pathways exist in humans. The extracellular signal-regulated protein kinase (ERK) pathway primarily transmits mitogenic and differentiation stimuli, while the c-Jun N-terminal kinase (JNK) and p38 pathways predominantly transmit stress and cytokine stimuli. ATF-2 (activating transcription factor 2, also known as CREB2 or cAMP responsive element binding protein 2) is a member of the ATF/CREB transcription factor family that binds to the cAMP response element (CRE). ATF-2 is activated by three MAPK pathways, ERK1/2, JNK and p38. Phosphorylation of Thr71 by MAPK14 or JNK increases transcriptional activity. MEF2, a member of the MADS box family, is mainly involved in muscle differentiation and is activated by both the p38 and ERK5 pathways. 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. c-Myc is activated by the MAP Kinase ERK1/2. c-Jun is a member of the activator protein-1 (AP-1) family and is activated by both ERK1/2 and JNK pathways. Jun proteins can also bind to form transcriptionally active heterodimers with CREB/ATF members. STAT1α is involved in the activation of IFNα and γ genes, and is activated by both the p38 and JNK pathways.
Figure 1: TransAM profiling of MAPK-regulated transcription factors.
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).
Figure 1: Flow chart of the TransAM process.
Contents & Storage
Two MAPK Family 96-well plates with plate sealer(s), phosphorylated ATF-2, phosphorylated c-Jun, c-Myc, MEF2 and STAT1α primary antibody(ies), 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.