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Transcription Factor Phosphorylation ELISA Kits

Phospho-Specific Transcription Factor Activity Assays

The Assay Genie Transcription Factor Activity Assay Kits are a premium range of Enzyme Linked Immunosorbent Assay (ELISA) kits used for the detection of active transcription factors in eukaryotic nuclear or cell lysates. Additionally, they may also be used to used to study the effects of phosphorylation on transcription factor activation. Determination of transcription factor activation is achieved via indirect ELISA methods.

Key Phospho-Specific Transcription Factor Activity Assay Kits


Features

  • Fast procedure - Significant reduction in runtime compared with other methods such as EMSA, Western Blot etc.

  • Easy-to-use - Simple ELISA format means rapid results versus other common methods of detecting active transcription factors.

  • Safer - Eliminates the need for harmful radioactive labelling.

  • High throughput - 96-well format allows lots of samples to be run simultaneously compared to gel shift assay methods.

Contents

  • Binding Buffer
  • Primary Antibody Diluent
  • Phospho-Specific Primary Antibody
  • Nuclear Wash Buffer
  • HRP-conjugated Secondary Antibody
  • Cytoplasmic Extraction Buffer
  • Nuclear Extraction Buffer
  • Nuclear Lysate Positive Control
  • Wild-Type Consensus dsDNA Oligonucleotide
  • Mutant Consensus dsDNA Oligonucleotide
  • Ready-to-use Substrate
  • Wash Buffer
  • Stop Solution

Applications

  • Detection and qualitative analysis of activated transcription factors in nuclear and cell lysates.

  • Establishment of the effects of phosphorylation on transcription factor activation.

  • Screening of compounds that interfere with DNA binding.

  • Signal transduction pathway analysis.

  • Establishment of target candidates in drug development.

Principle

The Assay Genie Phospho-Specific Transcription Factor Activity Assay (ELISA) Kit contains components necessary for the detection and analysis of active phosphorylated transcription factors in eukaryotic nuclear or cell lysates. This particular immunoassay utilizes the qualitative technique of an indirect ELISA.

Streptavidin is bound to the immunoassay plate and specific biotinylated double stranded (dsDNA) oligonucleotides are then added to bind to the streptavidin via a high affinity biotin-streptavidin interaction.

After subsequent blocking of extraneous binding sites in each well, the sample containing the target of interest can be added. Primary antibody is added to bind active phosphorylated transcription factors bound to the dsDNA oligonucleotide, which has been immobilized via the plate coated streptavidin.

A HRP-conjugated secondary antibody is added, which allows for specific binding to the Primary Antibody, and consequently colorimetric detection upon addition of the TMB (3, 3’, 5, 5’-Tetramethylbenzidine) substrate.

After addition of the substrate, a peroxidase catalysed reaction will produce a blue TMB Diimine product that is proportional to the target concentration in the sample. Colour development is quenched by addition of Stop Solution, or 2 N Sulfuric Acid, which turns the solution yellow.

The absorbance can then be read by a spectrophotometer at 450 nm and subsequently allowing for determination of the target concentration in the sample.

View a Transcription Factor Activity Assay Sample Protocol here.

Figure: Schematic representation of Assay Genie Phospho-Specific Transcription Factor Activity Assay principle


Key Phospho-Specific Transcription Factor Activity Assay Kits

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