Human c-Myc Transcription Factor Activity Assay

RayBio® Human c-Myc Transcription Factor Activity Assay. This assay uses a dsDNA coated plate with canonical c-Myc binding sequences to semi-quantitatively detect active c-Myc in lysates or nuclear extracts. Dry ice shipment (additional fee).

$402.00  
TFEH-CMYC-1
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Antigen Information

Accession Number:
  • P01106
Gene ID:
  • 4609
Gene Symbols:
  • BHLHE39
  • MYC
Protein Name & Synonyms:
Myc proto-oncogene protein, Class E basic helix-loop-helix protein 39 (bHLHe39), Proto-oncogene c-Myc, Transcription factor p64
Target Species:
  • Human

Assay Format

Pathway:
  • HER/ErbB Signaling
  • IGF Signaling
  • JAK/STAT Signaling
  • MAPK Signaling
  • Notch Signaling
  • Wnt/beta-Catenin Signaling
Specificity:
The olionucleotide/antibody pair provided in this kit recognizes human TFEB in whole lysates and nuclear extracts.
Number of Targets Detected:
1
Species Detected:
  • Human
Compatible Sample Types:
  • Cell Lysates
  • Nuclear Extracts
Design Principle:
  • Sandwich-based
Method of Detection:
Colorimetric
Quantitative/Semi-Quantitative:
  • Semi-Quantitative
Solid Support:
96-well Microplate

Product Specifications

Size:
1, 2, or 5 x 96-Well Strip Microplate Kit

Introduction

c-Myc is a transcription factor that belongs to the Myc family of proteins consisting of c-, N-, and L-Myc. Myc proteins are members of the basic helix-loop-helix (bHLH) protein family, in which the HLH domain is responsible for dimerization and the adjacent basic region mediates sequence-specific DNA binding. Myc has generally been associated with the promotion of cellular growth and proliferation, desensitization to growth-inhibitory stimuli, blockade of cell differentiation, cellular immortalization, and oncogenic transformation, as well as sensitization to apoptosis-inducing signals. Myc has also been linked to the regulation of various metabolic pathways, the induction of DNA damage, and genomic instability. In normal cells, c-Myc expression is generally induced by mitogens and suppressed by growth-inhibitory signals. Oncogenic activation of c-Myc occurs by direct gene alterations, such as translocation or amplification, or by mutations in upstream signaling pathways. These accidents commonly result in deregulated and/or elevated expression of c-Myc and its product. The activated c-Myc protein regulates multiple targeted genes through binding at the consensus DNA sequence CACGTG or E box.

Product Features

  • Specific transcription factor-DNA binding assay
  • Perfect alternative to EMSA
  • Easy to perform in an ELISA format
  • Non-radioactive assay
  • High throughput (96-well plate format)
  • Assay can be completed within 5 hours

Application Notes

Kit Components
  • 96-well Strip Microplate pre-coated with DNA probes
  • DNA Binding Buffer
  • Positive Control Sample
  • Specific Competitor DNA probe
  • Non-specific Competitor DNA probe
  • Assay Reagent
  • DTT
  • Wash Buffer
  • Primary Antibody
  • HRP-conjugated Secondary Antibody
  • Antibody Diluent Buffer
  • TMB One-Step Substrate Reagent
  • Stop Solution
Other Materials Required
  • Distilled or deionized water
  • 100 ml and 1 liter graduated cylinders
  • Tubes to prepare sample dilutions
  • Absorbent paper
  • Precision pipettes to deliver 2 µl to 1 ml volumes
  • Adjustable 1-25 ml pipettes for reagent preparation
  • Microplate reader capable of measuring absorbance at 450 nm
Protocol Outline
  1. Prepare all reagents and samples as instructed in the manual.
  2. Add 100 µl of sample or positive control to each well.
  3. Incubate 2 h at RT or O/N at 4 °C.
  4. Add 100 µl of prepared primary antibody to each well.
  5. Incubate 1 h at RT.
  6. Add 100 µl of prepared HRP-secondary antibody to each well.
  7. Incubate 1 h at RT.
  8. Add 100 µl of TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 µl of Stop Solution to each well.
  11. Read at 450 nm immediately.

Typical Data

Figure 1
Transcription factor assay of c-Myc from nuclear extracts of Jurkat cells or U937 cells. A. Western-blot result of c-Myc from cytoplasm and nuclear fractions. B. Transcription factor assay of c-Myc from nuclear fractions with the RayBio® c-Myc TF Activity Assay.

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Figure 2
Transcription factor assay of c-Myc from nuclear extracts of Jurkat cells or U937 cells with the specific competitor or non-specific competitor. The result shows specific binding of c-Myc to the DNA binding site.

Storage/Stability

Upon receipt, the positive control should be removed and stored at -20° or -80°C. The remainder of the kit can be stored for up to 6 months at 2-8°C from the date of shipment. Opened Microplate Wells or reagents may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge.
Note: The kit can be used within one year if the whole kit is stored at -20°C upon receipt. Avoid repeated freeze-thaw cycles.
It works good on the human Burkitt lymphoma cell lysate, incubation time is short so we get the result quickly.
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Usage

This product is furnished for LABORATORY RESEARCH USE ONLY.
Not for diagnostic or therapeutic use.

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