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    ap1-c-fosfosbfra1c-junjund-transcription-factor-assay-kit-colorimetric-ab207196.pdf

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Epigenetics and Nuclear Signaling Transcription Domain Families HLH / Leucine Zipper Leucine Zipper
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AP1 (c-Fos/FosB/Fra1/c-Jun/JunD) Transcription Factor Assay Kit (Colorimetric) (ab207196)

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  • SDS
  • Protocol Booklet
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Nuclear extracts from K-562 cells stimulated with TPA were assayed for activity of AP1 family members c-Jun, c-Fos, FosB, JunB, JunD and Fra-1.

    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

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    Overview

    • Product name

      AP1 (c-Fos/FosB/Fra1/c-Jun/JunD) Transcription Factor Assay Kit (Colorimetric)
    • Detection method

      Colorimetric
    • Sample type

      Nuclear Extracts
    • Assay type

      Semi-quantitative
    • Sensitivity

      < 1250 ng/well
    • Assay time

      3h 30m
    • Species reactivity

      Reacts with: Human
    • Product overview

      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.

    • Notes

      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.

    • Platform

      Microplate reader

    Properties

    • Storage instructions

      Please 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
    • Research areas

      • Epigenetics and Nuclear Signaling
      • Transcription
      • Domain Families
      • HLH / Leucine Zipper
      • Leucine Zipper
      • Epigenetics and Nuclear Signaling
      • Transcription
      • Cancer susceptibility
      • Proto-oncogenes
      • Epigenetics and Nuclear Signaling
      • Transcription
      • Transcription Factors
    • Cellular localization

      JunB: 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.
    • Database links

      • Entrez Gene: 2353 Human
      • Entrez Gene: 3726 Human
      • Entrez Gene: 8061 Human
      • Omim: 136515 Human
      • Omim: 165161 Human
      • SwissProt: P01100 Human
      • SwissProt: P05412 Human
      • SwissProt: P15407 Human
      • SwissProt: P17275 Human
      • SwissProt: P17535 Human
      • SwissProt: P53539 Human
      • Unigene: 25292 Human
      • Unigene: 25647 Human
      • Unigene: 283565 Human
      see all

    Associated products

      Images

      • Nuclear extracts from K-562 cells stimulated with TPA were assayed for activity of AP1 family members c-Jun, c-Fos, FosB, JunB, JunD and Fra-1.
        Nuclear extracts from K-562 cells stimulated with TPA were assayed for activity of AP1 family members c-Jun, c-Fos, FosB, JunB, JunD and Fra-1.

        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

      Protocols

      • Protocol Booklet

      Click here to view the general protocols

      Datasheets and documents

      • Datasheet
      • SDS
    • References (2)

      Publishing research using ab207196? Please let us know so that we can cite the reference in this datasheet.

      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

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