Overview

  • Product name

    MyoD Transcription Factor Assay Kit (Colorimetric)
  • Detection method

    Colorimetric
  • Sample type

    Nuclear Extracts
  • Assay type

    Semi-quantitative
  • Sensitivity

    150 ng/well
  • Assay time

    3h 30m
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Product overview

    MyoD Transcription Factor Assay Kit (Colorimetric) (ab207214) is a high throughput assay to quantify MyoD 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 MyoD consensus binding site (5’ –CACCTG– 3’) has been immobilized onto a 96-well plate. Active MyoD present in the nuclear extract specifically binds to the oligonucleotide. MyoD is detected by a primary antibody that recognizes an epitope of MyoD 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 MyoD.


    Key performance and benefits:



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

    • Detection limit: < 0.15 µg nuclear extract/well.

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

  • Notes

    The Myogenic Regulatory Factor (MRF) family is composed of 4 members:  Myf-3 (MyoD), Myf-4 (myogenin), Myf-5 and Myf-6 (MRF4/herculin). These proteins are involved in the skeletal muscle differentiation program that balances myoblast proliferation vs. differentiation. Activation of this program in response to growth factor withdrawal is accompanied by myoblast cell fusion to form multinucleated myotubes, irreversible commitment of muscle cells to the post-mitotic state, and transcriptional activation of muscle-specific genes. MyoD is expressed in undifferentiated myogenic proliferating cells. Expression of MyoD family genes activates myogenesis in various non-muscle cell types. In mice bearing a null Myf-5/MyoD double mutation, muscle fibers and myoblasts are absent. MyoD acts downstream of Pax-3 and Myf-5 in skeletal myogenesis and is sensitive to growth factors and transforming gene products such as Ras and Fos, which have been shown to abolish the expression of MyoD in myoblasts. MyoD induces muscle-specific alternative splicing of mouse mitochondrial ATP synthase gamma-subunit, beta-tropomyosin, MEF2A and MEF2D pre-mRNAs.

    MyoD is a transcription factor with a basic helix-loop-helix (bHLH) DNA-binding domain that mediates dimerization and binding to a DNA consensus sequence termed an E-box (CANNTG). These E-boxes are present in the control regions of several skeletal muscle-specific genes such as the muscle creatinine kinase, myosin light chains 1 and 3, desmin and the acetylcholine receptor. Its dimeric partners are E-box binding proteins such as Tal-1, E12 and E476. While MyoD and E47 both form homodimeric complexes with DNA that have similar dissociation constants, a stochiometric mixture of these bHLH domains forms almost exclusively heterodimeric complexes on DNA. The DNA binding activity of MyoD is repressed by forming heteromeric complexes with inhibitory proteins, such as Id protein and Mist1 transcription factor, which occupy the specific E-box target sites.

  • 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 MyoD 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
    C2C12 nuclear extract (2.5 μg/μL) 1 x 40µl 1 x 200µl
    Developing Solution 1 x 11ml 1 x 55ml
    Dithiothreitol (DTT) (1 M) 1 x 100µl 1 x 500µl
    Lysis Buffer 1 x 10ml 1 x 50ml
    MyoD antibody (0.2 μg/μL) 1 x 11µl 1 x 55µl
    MyoD mutated oligonucleotide (10 pmol/μL) 1 x 100µl 1 x 500µl
    Plate sealer 1 unit 5 units
    Protease Inhibitor Cocktail 1 x 100µl 1 x 500µl
    Stop Solution 1 x 11ml 1 x 55ml
    Wild-type oligonucleotide (10 pmol/μL) 1 x 100µl 1 x 500µl
  • Research areas

  • Relevance

    Myogenin D1 belongs to the basic helix-loop-helix family of transcription factors and the myogenic factors subfamily. It is involved in muscle cell differentiation, and is essential for repair of damaged tissue. It activates its own transcription which may stabilize commitment to myogenesis.
  • Cellular localization

    Nuclear
  • Alternative names

    • MYF 3
    • MYF3
    • MYO D1
    • Myoblast determination protein 1
    • MYOD
    • MYOD 1
    • MYOD1
    • Myogenic differentiation 1
    • Myogenic factor 3
    • MyogeninD1
    • PUM
    see all
  • Database links

Images

  • Different amounts of unstimulated NIH 3T3 (light grey) and C2C12 (dark grey) were tested for MyoD activation. These results are provided for demonstration purposes only.

Protocols

References

ab207214 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

There are currently no Customer reviews or Questions for ab207214.
Please use the links above to contact us or submit feedback about this product.

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com

Sign up