Transcription Factor Activity Assay FAQs

1. 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.

2. 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

3. 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 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

4. 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