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Transcription Factor ELISA - Protocol

The TFact DNA-Binding ELISA utilizes an indirect ELISA method to facilitate the detection and qualitative determination of active transcription factor profiles in a variety of nuclear and cell lysates. Methods such as electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitation (ChIP), Western Blotting (WB) and reporter gene fusion are often time-consuming and complicated.

Introduction:

The TFact DNA-Binding ELISA Kit contains components necessary for detection of active 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 activated transcription factors bound to the dsDNA oligonucleotide, which has been immobilized via the plate-coated streptavidin. A HRP-conjugated secondary antibody specific for rabbit IgGs is added, which allows for specific binding to the Primary Antibody, and consequently colorimetric detection upon addition of the TMB substrate.

For color development, TMB (3, 3’, 5, 5’-Tetramethylbenzidine) is added to each well. After addition of the substrate, a peroxidase catalyzed reaction will produce a blue TMB Diimine product that is proportional to the target concentration in the sample. Color 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.

Materials (Phospho TFact):

  • Reagent
  • 96-Well dsDNA Oligonucleotide Coated Microplate
  • 100x Primary Anitbody
  • 100x Primary Phospho Antibody
  • HRP-Conjugated Anti-Rabbit IgG Antibody
  • Nuclear Lysate Positive Control
  • Wild-Type Consensus dsDNA Oligonucleotide
  • Mutant Consensus dsDNA Oligonucleotide
  • 10x Wash Buffer
  • 2x Binding Buffer
  • Primary Antibody Diluent
  • Nuclear Wash Buffer
  • Cytoplasmic Extraction Buffer
  • Nuclear Extraction Buffer
  • Ready-to-use Substrate
  • Stop Solution
  • Adhesive Plate Sealers


  • Quantity
  • 12 x 8-Well Microstrips
  • 100 μl (100x)
  • 100 μl (100x)
  • 12 ml (1x)
  • Varies (Lyophilized)
  • 20 μl
  • 20 μl
  • 50 ml (10x)
  • 12 ml (2x)
  • 12 ml (1x)
  • 12 ml (1x)
  • 6 ml (1x)
  • 6 ml (1x)
  • 12 ml (1x)
  • 12 ml (1x)
  • 2 Sheets

Assay Protocol:

1. The Nuclear Lysate Positive Control is lyophilized; reconstitute by adding 110 μl of 1x Binding Buffer. It is advised to run the positive control in duplicate or triplicate. The suggested dilutions for Nuclear Lysate Positive Control in 1x Binding Buffer are 1:10, 1:20, 1:40, and Blank.

2. Add 100 μl of Nuclear Lysate Positive Control dilutions to the appropriate wells. For the negative Nuclear Lysate Positive Control well, add 100 μl of 1x Binding Buffer.

3. In the Primary Antibody and Primary Phospho-Antibody negative controls, the Primary Antibody and Primary Phospho Antibody are left out to correct for any background noise. The Primary Antibody negative controls should be performed for both the Nuclear Lysate Positive Control and samples. Follow the volumes below for Primary Antibody negative controls for the Nuclear Lysate Positive Controls.

4. Add 100 μl of Nuclear Lysate Positive Control dilutions or Sample dilutions to the appropriate Primary Antibody or Primary Phospho-Antibody negative control wells. For the negative Nuclear Lysate Positive Control and negative Sample wells, add 100 μl of 1x Binding Buffer.

Optional:
5. We recommend a final concentration of 0.5 nmol of Wild-Type (WT Oligo) or Mutant (MT Oligo) Oligonucleotide in each well. The suggested dilutions for the Wild-Type Oligonucleotide Control follow the recommended positive control with addition of 2 μl of WT Oligo in each Nuclear Lysate Positive Control working solution.

6. Add 100 μl of WT Oligo Control Dilution into the appropriate WT Oligo Control wells.

7. The suggested dilutions for the MT Oligo Control follow the recommended positive control with addition of 2 μl of MT Oligo in each positive control.

8. Add 100 μl of MT Oligo Control Dilution into the appropriate MT Oligo Control wells.

Continued at this step with or without optional WT and or MT addtion:

9. Determine the volume and dilution necessary for your application. Using 2x Binding Buffer, add appropriate volume so that the final working Sample Dilution contains 1x Binding Buffer.

10. Add 100 μl of diluted samples to corresponding wells. For negative sample wells, add 100 μl of 1x Binding Buffer. Incubate plate on orbital shaker at room temperature for 2 hours.

11. Wash 3 times with 1x Wash Buffer with gentle shaking in-between.

12. Add 100 μl of working Primary Antibody or Primary Phospho-Antibody solution to every well that is being used except the Primary Antibody or Primary Phospho-Antibody negative controls for Nuclear Lysate Positive Controls and samples. For the Primary Antibody or Primary Phospho-Antibody negative controls, add 100 μl of Primary Antibody Diluent. Leave the on orbital shaker at room temperature for 2 hours.

13. Wash 3 times with 1x Wash Buffer with gentle shaking in-between.

14. Add 100 μl of HRP-Conjugated Anti-Rabbit IgG Antibody to each well that is being used. Incubate on orbital shaker at room temperature for 1 hour.

15. Wash 3 times with 1x Wash Buffer with gentle shaking in-between.

16. Add 50 μl of Ready-to-Use Substrate to every well that is being used. Keep those wells away from light and leave on orbital shaker for 10 to 30 minutes until there is distinctive blue color development from the wells. Closely monitor color development as some wells may develop faster than others. The reaction should be terminated when the well with greatest blue color ceases to continue developing.

17. When color development is sufficient, add 100 μl of Stop Solution to each well that is being used. Leave on orbital shaker for 1 minute or shake by hand to ensure color development is completely stopped. There will be a noticeable color change from blue to yellow.

18. The plate is now ready to read. Within 30 minutes of adding Stop Solution, determine the optical density or absorbance of each well by reading on a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from readings at 450 nm.