Overview

  • Product name
    c-Myc Transcription Factor Assay Kit (Colorimetric)
  • Detection method
    Colorimetric
  • Sample type
    Nuclear Extracts
  • Assay type
    Semi-quantitative
  • Sensitivity
    < 250 ng/well
  • Assay time
    3h 30m
  • Species reactivity
    Reacts with: Mouse, Human
  • Product overview

    c-Myc Transcription Factor Assay Kit (Colorimetric) (ab207200) is a high throughput assay to quantify c-Myc activation in nuclear extracts. 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 c-Myc consensus binding site (5’ –CACGTG– 3’) has been immobilized onto a 96-well plate. Active c-Myc present in the nuclear extract specifically binds to the oligonucleotide. c-Myc is detected by a primary antibody that recognizes an epitope of c-Myc accessible only when the protein is activated and bound to its target DNA. An HRP-conjugated secondary antibody provides sensitive colorimetric readout that at OD 450 nm. This product detects human and mouse c-Myc.


    Key performance and benefits:



    • Assay time: 3.5 hours (cell extracts preparation not included).

    • Detection limit: < 0.25 µg nuclear extract/well.

    • Detection range: 0.25 – 5 µg nuclear extract/well.

  • Notes

    c-Myc is a transcription factor that regulates cell growth and differentiation, glycolysis and apoptosis and deregulation of c-Myc has been implicated in the origin of diverse human cancers.

    c-Myc was originally discovered as the cellular homolog of the retroviral v-myc oncogene, and is a transcription factor involved in a wide variety of cellular processes, including cell proliferation, replicative potential, growth, differentiation and apoptosis. Expression of c-Myc is induced by mitogenic signals and suppressed by growth-inhibitory signals. c-Myc is a member of the basic helix-loop-helix leucine zipper (bHLHzip) family, along with B-Myc, N-Myc, L-Myc and s-Myc. Upon dimerization with the bHLHzip protein Max, c-Myc can bind to the E box motif CACGTG and activate transcription. c-Myc is phosphorylated at Ser62, which has been shown to be a regulatory site of phosphorylation. The phosphorylation of c-Myc causes increased function of the NH2-terminal transactivation domain, and studies have indicated that the expression of MAP kinase is responsible for increased c-Myc phosphorylation at Ser62.

    Because of the central role of c-Myc as an activator of diverse cellular processes, regulation of this transcription factor is crucial for proper cell function and ultimate survival. The main regulation of c-Myc occurs through its binding with the bHLHzip protein Max, which can also form heterodimers with members of the Mad family (Mad 1, 3, 4 and Mxi1). As c-Myc cannot bind to DNA without forming a heterodimer with Max, competition between c-Myc and Mad for the common partner Max is used to regulate c-Myc activity. While Max is a relatively stable protein, c-Myc degrades rapidly, with a half-life of 20-30 minutes.

  • Platform
    Microplate reader

Properties

  • Storage instructions
    Please refer to protocols.
  • Components 1 x 96 tests 5 x 96 tests
    10X Antibody Binding Buffer 1 x 2.2ml 1 x 11ml
    10X Wash Buffer 1 x 22ml 1 x 110ml
    96-well c-Myc assay plate 1 unit 5 units
    Anti-rabbit HRP-conjugated IgG (0.25 μg/μL) 1 x 11µl 1 x 55µl
    Binding Buffer 1 x 10ml 1 x 50ml
    c-Myc antibody 1 x 11µl 1 x 55µl
    c-Myc Mutated oligonucleotide (10 pmol/μL) 1 x 100µl 1 x 500µl
    c-Myc Wild-type oligonucleotide (10 pmol/μL) 1 x 100µl 1 x 500µl
    Developing Solution 1 x 11ml 1 x 55ml
    Dithiothreitol (DTT) (1 M) 1 x 100µl 1 x 500µl
    Jurkat (1 day growth) nuclear extract (2.5 μg/μL) 1 x 40µl 1 x 200µl
    Lysis Buffer 1 x 10ml 1 x 50ml
    Plate sealer 1 unit 5 units
    Protease Inhibitor Cocktail 1 x 100µl 1 x 500µl
    Stop Solution 1 x 11ml 1 x 55ml
  • Research areas
  • Function
    Participates in the regulation of gene transcription. Binds DNA in a non-specific manner, yet also specifically recognizes the core sequence 5'-CAC[GA]TG-3'. Seems to activate the transcription of growth-related genes.
  • Involvement in disease
    Note=Overexpression of MYC is implicated in the etiology of a variety of hematopoietic tumors.
    Note=A chromosomal aberration involving MYC may be a cause of a form of B-cell chronic lymphocytic leukemia. Translocation t(8;12)(q24;q22) with BTG1.
    Defects in MYC are a cause of Burkitt lymphoma (BL) [MIM:113970]. A form of undifferentiated malignant lymphoma commonly manifested as a large osteolytic lesion in the jaw or as an abdominal mass. Note=Chromosomal aberrations involving MYC are usually found in Burkitt lymphoma. Translocations t(8;14), t(8;22) or t(2;8) which juxtapose MYC to one of the heavy or light chain immunoglobulin gene loci.
  • Sequence similarities
    Contains 1 basic helix-loop-helix (bHLH) domain.
  • Post-translational
    modifications
    Phosphorylated by PRKDC. Phosphorylation at Thr-58 and Ser-62 by GSK3 is required for ubiquitination and degradation by the proteasome.
    Ubiquitinated by the SCF(FBXW7) complex when phosphorylated at Thr-58 and Ser-62, leading to its degradation by the proteasome. In the nucleoplasm, ubiquitination is counteracted by USP28, which interacts with isoform 1 of FBXW7 (FBW7alpha), leading to its deubiquitination and preventing degradation. In the nucleolus, however, ubiquitination is not counteracted by USP28, due to the lack of interaction between isoform 4 of FBXW7 (FBW7gamma) and USP28, explaining the selective MYC degradation in the nucleolus. Also polyubiquitinated by the DCX(TRUSS) complex.
  • Cellular localization
    Nucleus > nucleoplasm. Nucleus > nucleolus.
  • Information by UniProt
  • Alternative names
    • AU016757
    • Avian myelocytomatosis viral oncogene homolog
    • bHLHe39
    • c Myc
    • Class E basic helix-loop-helix protein 39
    • MRTL
    • Myc
    • Myc protein
    • Myc proto oncogene protein
    • Myc proto-oncogene protein
    • myc-related translation/localization regulatory factor
    • MYC_HUMAN
    • Myc2
    • MYCC
    • Myelocytomatosis oncogene
    • Niard
    • Nird
    • Oncogene Myc
    • OTTHUMP00000158589
    • Proto-oncogene c-Myc
    • Protooncogene homologous to myelocytomatosis virus
    • RNCMYC
    • Transcription factor p64
    • Transcriptional regulator Myc-A
    • V-Myc avian myelocytomatosis viral oncogene homolog
    • v-myc myelocytomatosis viral oncogene homolog (avian)
    see all
  • Database links

Images

  • Different amounts of unstimulated U-937 (grey) and Jurkat (black) were tested for c-Myc activation. These results are provided for demonstration purposes only.

Protocols

References

ab207200 has not yet been referenced specifically in any publications.

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