Transcription Factor Activity Assay (ELISA) Kits

Transcription Factor Activity Assays

The ELISA 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.

ELISA Genie Transcription Factor Activity Assay Kits allow scientists to perform high throughput screening of hundreds of samples in a convenient, efficient, and sensitive manner. This accelerates the journey from research to publication or development of a new drug.

Key ELISA Genie Transcription Factor Activity Assays


Advantages of ELISA Genie Transcription Factor Activity Assay Kits

  • Quicker, less complicated, and easier to achieve satisfactory results than common methods of detection of transcription factor binding to DNA elements and motifs.
  • Significant reduction in runtime in comparison to common methods of detection of transcription factor binding to DNA elements and motifs - an ELISA Genie Transcription Factor Activity Assay can be completed in less than one day.
  • Eliminates the need for harmful radioactive labeling.
  • High sensitivity and signal-to-noise ratio.

Applications of ELISA Genie Transcription Factor Activity Assay Kits

  • Detection and qualitative analysis of activated transcription factors in nuclear and cell lysates.
  • Signal transduction pathway analysis.
  • Establishment of target candidates in drug development.

How do ELISA Genie Transcription Factor Activity Assay Kits work?

The ELISA Genie Transcription Factor Activity Assay (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 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 ELISA Genie Transcription Factor Activity Assay principle


Contents of ELISA Genie Transcription Factor Activity Assay Kits

Component Quantity Component Quantity

Binding Buffer

12 ml

96-Well dsDNA Oligonucleotide Coated Microplate

12 x 8-Well
Microstrips

Primary Antibody Diluent

12 ml

Primary Antibody/Primary Phospho-Antibody

100 μl

Nuclear Wash Buffer

12 ml

HRP-conjugated Secondary Antibody

2 x 6 ml

Cytoplasmic Extraction Buffer

6 ml

Nuclear Lysate Positive Control

Lyophilized

Nuclear Extraction Buffer

6 ml

Wild-Type Consensus dsDNA Oligonucleotide

20 μl

Ready-to-Use Substrate

12 ml

Mutant Consensus dsDNA Oligonucleotide

20 μl

Stop Solution

12 ml

Wash Buffer

50 ml


Storage - Transcription Factor Activity Assay Kits

If samples are ready to be used within 24 hours, aliquot and store at 4°C. If samples are to be saved for future or long term use, aliquot into multiple tubes and store at -80°C. Avoid repeated freeze/thaw cycles to prevent loss of biological activity of transcription factors in experimental samples.


Assay Restrictions of ELISA Genie Transcription Factor Activity Assay Kits

ELISA Genie Transcription Factor Activity Assay Kits are intended for research purposes only, NOT diagnostic or clinical procedures of any kind.

Materials included in ELISA Genie Transcription Factor Activity Assay Kits should NOT be used past the expiration date on the kit label.

Reagents or substrates included in this kit should NOT be mixed or substituted with reagents or substrates from any other kits.

Variations in pipetting technique, washing technique, operator laboratory technique, kit age, incubation time or temperature may cause differences in binding affinity of the materials provided.

The assay is designed to eliminate interference and background by other cellular macromolecules or factors present within any biological samples. However, the possibility of background noise cannot be fully excluded until all factors have been tested using the assay kit.


Key ELISA Genie Transcription Factor Activity Assay Kits


FAQs for ELISA Genie Transcription Factor Activity Assay Kits  

1. How many samples can I test on one ELISA Genie Transcription Factor Activity Assay Kits?

On each 96 well plate, an 8-point standard curve is run with duplicate wells. The remaining wells may then be used for samples (duplicates are strongly recommended). If the entire plate is used in 1 experiment, the remaining 80 wells will accommodate 40 samples in duplicate. If not run all at once, a new standard curve must be included with every run, reducing the number of potential samples by 8 (to account for the new standard curve points).

2. My kit was left out at room temperature for several days. Can it still be used?

The kit may have diminished activity after an extended period at room temperature, but it is most likely still usable, especially if left out for only a small amount of time. The most temperature-sensitive components in the kit are the standards, HRP-streptavidin, and detection antibody.

3. Why is there a weak signal/no signal detected from my ELISA Genie Transcription Factor Activity Assay?

This may due to a number of reasons. The possible causes and their solutions are listed below:

Possible cause Possible solution

Incorrect nuclear lysate

Choose different cell line

Incorrect lysate preparation or storage

Add protease and phosphatase inhibitors, keep everything on ice, and store at -80°C and avoid freeze/thaw cycles

Key reagents missing

Consult manual and ensure all steps are followed

Incorrect volume of reagents added

Consult manual and ensure all steps are followed

Incorrect storage of plate and/or reagents

Keep everything at specific temperature

4. Why is their high background noise from my ELISA Genie Transcription Factor Activity Assay?

This may be for a number of reasons. The possible causes and their solutions are listed below:

Possible cause Possible solution

Inadequate washing between steps

Ensure the proper volume of wash buffer and steps

Too much primary or secondary antibody

Reduce concentration

Buffer/Reagent contamination

Ensure sterile techniques are used to maintain quality of reagents

Too much nuclear lysate

Use higher dilutions

Too much substrate

Reduce substrate used

Substrate Reagent incubation time is too long

Reduce incubation time until adequate color development

5. Why may the colour development of my ELISA Genie Transcription Factor Activity Assay be uneven?

This may be for a number of reasons. The possible causes and their solutions are listed below:

Possible cause Possible solution

Inadequate washing between steps

Ensure the proper volume of wash buffer and steps

Incorrect order or location in addition of reagents steps

Use template provided and ensure protocol is strictly followed

Cross contamination

Use sterile technique  

Uneven reagent addition or washing of wells

Ensure multi-channel pipette or plate washer is calibrated and not clogged


Health & Safety Precautions of ELISA Genie Transcription Factor Activity Assay Kits

The contents of ELISA Genie Transcription Factor Activity Assay Kits should be handled by those trained in laboratory techniques and used according to the principles of good laboratory practice.

Reagents provided in ELISA Genie Transcription Factor Activity Assay Kits may be harmful if ingested, inhaled or absorbed through the skin. Please carefully review the MSDS for each reagent before conducting the experiment.

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