Transcription Factor Activity Assay Kits
Transcription Factor Assays
Detect Transcription Factors via indirect ELISA methods!
Detect transcription factors or study transcription factor phosphorylation!
Transcription Factor Assay principle. Click to enlarge!
Transcription Factor Assay Features
- Simple | Measure Transcription Factor DNA-binding in 96-wells simultaneously
- Rapid | Significant reduction in runtime compared with other methods such as EMSA, Western Blot etc.
- Safe | Eliminates the need for harmful radioactive labelling
- High throughput | 96-well format allows many samples to be run simultaneously compared to gel shift assay methods
- Choice | Range of Transcription Factor and Phospho-specific Transcription Factor Assays available!
Transcription Factor Assay Applications
- Semi-quantitatively detect transcription factor-DNA complexes in nuclear and cell lysates
- Analyse signal transduction pathways
- Investigate target candidates in drug development
- Screen for inhibitors & activators of transcription factor activity
- Establishment of the effects of phosphorylation on transcription factor activation.
Phospho-specific Transcription Factor Assay principle. Click to enlarge!
Transcription Factor Assays
Transcription Factor Activity Assays
Phospho-Specific Transcription Factor Activity Assay
Transcription Factor Assay Principle
The Assay 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.
Transcription Factor Assay FAQs
At Assay Genie we have compiled a list of some commonly asked questions regarding Transcription Factor Assays.
TF Assay FAQs
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.
Why is there a weak signal/no signal detected from 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 |
Why is their high background noise from my Transcription Factor Activity Assay?
Possible Cause | Possible Solution |
Inadequate washing between steps |
Ensure the proper volume of wash buffer and adhere to the protocol carefully to ensure correct number of washing steps are completed |
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 |
This may be for a number of reasons. The possible causes and their solutions are listed below:
Why may the colour development of 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 |