Key features and details
- Assay type: Semi-quantitative
- Detection method: Colorimetric
- Platform: Microplate reader
- Assay time: 3 hr 30 min
- Sample type: Nuclear Extracts
- Sensitivity: 1250 ng/well
Product nameAP1 (c-Fos/FosB/Fra1/c-Jun/JunD) Transcription Factor Assay Kit (Colorimetric)
Sample typeNuclear Extracts
Sensitivity< 1250 ng/well
Assay time3h 30m
Species reactivityReacts with: Human
AP1 (c-Fos/FosB/Fra1/c-Jun/JunB/JunD) Transcription Factor Assay Kit (Colorimetric) (ab207196) is a high throughput assay to quantify activation of AP1 family members (c-Fos/FosB/Fra1/c-Jun/JunB/JunD) at the same time in one assay. This assay combines a quick ELISA format with a sensitive and specific non-radioactive assay for transcription factor activation.
A specific double stranded DNA sequence containing the TPA-responsive element (TRE) (5´ –TGAGTCA– 3´) has been immobilized onto a 96-well plate. Activator protein-1 (AP1) dimers present in the nuclear extract specifically bind to the oligonucleotide. The primary antibodies used recognize epitopes on c-Fos, FosB, Fra-1, c-Jun, JunB or JunD proteins accessible only when the protein is activated and bound to its target DNA. An HRP-conjugated secondary antibody provides sensitive colorimetric readout at OD 450 nm. This product detects human, mouse and rat c-Fos, FosB and JunD, human and mouse c-Jun and human Jun B and Fra-1.
Key performance and benefits:
- Assay time: 3.5 hours (cell extracts preparation not included).
- Detection limit: < 1.25 µg nuclear extract/well.
- Detection range: 0.1 – 20 µg nuclear extract/well.
The activator protein-1 (AP1) transcription factors belong to a large family of structurally related transcription factors that includes ATF1-4, c-Fos, c-Jun, c-Myc and C/EBP. The members of this family, named bZIP, share a dimerization domain with a leucine zipper motif and a DNA binding domain rich in basic residues (lysines and arginines). AP1 is composed of a mixture of heterodimeric complexes of proteins derived from the Fos and Jun families including c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB and JunD. Only Jun proteins can form transcriptionally active homodimers within AP1 members, or heterodimers with CREB/ATF members, to bind the CRE element (5´-TGACGTCA-3´). Primarily, AP1 dimers bind to DNA on a TPA-response element (TRE) with the 5´-TGA(C/G)TCA-3´ sequence. Jun-Fos heterodimers form more stable complexes with TREs. These complexes display stronger transactivating activity than Jun-Jun homodimers.
Phosphorylation of AP1 family members by kinases is required for transactivation activity. In the case of c-Jun, the activation domain is regulated to a large extent by the JNK family of MAP kinases. JNK kinases phosphorylate c-Jun at Ser-63, resulting in the binding of c-Jun to the CBP/p300 family of transcriptional co-activators. For the Fos proteins, both N- and C-terminal domains flanking the bZIP domain require phosphorylation for biological activity.
AP1 expression is induced by multiple stimuli such as serum, growth factors, phorbol esters and oncogenes. These include peptide growth factors, cytokines of the TGF beta, TNF, and interferon families, neuronal depolarization and cellular stress. Upon serum starvation of human fibroblast cells, Fos and Jun protein production can be induced for up to 4 hours by adding serum. Interestingly, serum starvation lowers basal expression of FosB and c-Fos but has no significant effect on c-Jun.
AP1 proteins play a role in the expression of many genes involved in proliferation and cell cycle progression including neuronal apoptosis, learning process, drug-induced behavorial responses, bone growth and differentiation, and embryo development. For instance, cell transformation by oncogenes that function in the growth factor signal transduction pathway, such as ras, rasF and mek, results in a high increase in AP1 component protein expression. Therefore, AP1-regulated genes support the invasive process observed during malignancy and metastasis.
Storage instructionsPlease refer to protocols.
Components 2 x 96 tests 10X Antibody Binding Buffer 2 x 2.2ml 10X Wash Buffer 1 x 60ml 96-well assay plate 2 units Anti-rabbit HRP-conjugated IgG (0.25 μg/μL) 2 x 11µl AP-1 Mutated oligonucleotide (10 pmol/μL) 1 x 100µl AP-1 Wild-type oligonucleotide (10 pmol/μL) 1 x 100µl Binding Buffer 1 x 10ml c-Fos antibodies 1 x 11µl Developing Solution 2 x 11ml Dithiothreitol (DTT) (1 M) 1 x 100µl FosB antibodies 1 x 11µl Fra-1 antibodies 1 x 11µl JunB antibodies 1 x 11µl JunD antibodies 1 x 11µl K-562(TPA) nuclear extract (2.5µg/μL) 1 x 40µl Lysis Buffer 1 x 10ml Phospho-c-Jun antibody 1 x 22µl Plate sealer 2 units Poly [d(l-c)] (17 µg/μL) 1 x 100µl Protease Inhibitor Cocktail 1 x 100µl Stop Solution 1 x 60ml
Cellular localizationJunB: Nucleus. FRA1: Nucleus. c-Fos+FosB+c-Jun+JunD: Nucleus. Endoplasmic reticulum. Cytoplasm, cytosol. In quiescent cells, present in very small amounts in the cytosol. Following induction of cell growth, first localizes to the endoplasmic reticulum and only later to the nucleus. Localization at the endoplasmic reticulum requires dephosphorylation at Tyr-10 and Tyr-30.
Nuclear extracts from K-562 cells stimulated with TPA (5 µg) were assayed for activity of AP1 family members c-Jun, c-Fos, FosB, JunB, JunD and Fra-1 in the absence (grey) or presence of wild-type (black) or mutated (white) consensus binding oligonucleotides. These results are provided for demonstration purposes only
ab207196 has been referenced in 2 publications.
- Yang N et al. Tumor necrosis factor-related apoptosis-inducing ligand additive with Iodine-131 of inhibits non-small cell lung cancer cells through promoting apoptosis. Oncol Lett 16:276-284 (2018). PubMed: 29928412
- Jang B et al. The Peptidylarginine Deiminase Inhibitor Cl-Amidine Suppresses Inducible Nitric Oxide Synthase Expression in Dendritic Cells. Int J Mol Sci 18:N/A (2017). PubMed: 29077055