Introduction
The RayBio® ATF-1 Transcription Factor Activity Assay Kit is a non-radioactive transcription factor assay with an ELISA format. This format offers an easy, speedy, sensitive, and high-throughput method to detect the activation of transcription factors. In 96-well plates, double stranded oligonucleotides containing ATF-1 binding sequence have been coated. These oligonucleotides specifically capture the active ATF-1 contained in whole cell lysate or nuclear extracts after a short incubation. Subsequently, the primary antibody against ATF-1 recognizes the ATF-1-DNA complex in each well, and an HRP-conjugated secondary antibody is then used for detection. After washing away any unbound antibody, signal can be obtained easily through a colorimetric assay with a spectrophotometric plate reader at 450 nm. The specificity of the reaction between active ATF-1 and the DNA probe is additionally stringent because of the establishment of specific competitive DNA and non-specific competitive DNA probes in this reaction system.
Product Features
- Specific transcription factor-DNA binding assay
- Perfect alternative to EMSA
- Easy to perform in an ELISA format
- Non-radioactive assay
- High throughput (96-well plate format)
- Assay can be completed within 5 hours
Application Notes
Kit Components
- 96-well Strip Microplate pre-coated with DNA probes
- DNA Binding Buffer
- Positive Control Sample
- Specific Competitor DNA probe
- Non-specific Competitor DNA probe
- Assay Reagent
- DTT
- Wash Buffer
- Primary Antibody
- HRP-conjugated Secondary Antibody
- Antibody Diluent Buffer
- TMB One-Step Substrate Reagent
- Stop Solution
Other Materials Required
- Distilled or deionized water
- 100 ml and 1 liter graduated cylinders
- Tubes to prepare sample dilutions
- Absorbent paper
- Precision pipettes to deliver 2 µl to 1 ml volumes
- Adjustable 1-25 ml pipettes for reagent preparation
<lIκBenchtop rocker or shaker
- Microplate reader capable of measuring absorbance at 450 nm
Protocol Outline
- Prepare all reagents and samples as instructed in the manual.
- Add 100 µl of sample or positive control to each well.
- Incubate 2 h at RT or O/N at 4°C.
- Add 100 µl of prepared primary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of prepared HRP-secondary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of TMB One-Step Substrate Reagent to each well.
- Incubate 30 min at RT.
- Add 50 µl of Stop Solution to each well.
- Read at 450 nm immediately.
Typical Data
Figure 1
Transcription Factor assay of ATF-1 detects activated ATF-1 from 293T cell nuclear fractions. The 293T cells were treated with 2ug/mL Tunicamycin in 2% FBS D-MEM for 8 hours and were then collected.
Figure 2
Transcription factor assay of ATF-1 from nuclear extracts of 293T cells treated with Tunicamycin with the specific competitor or non-specific competitor. The result shows specific binding of ATF-1 to the conserved binding site detected by using the RayBio® ATF-1 TF Activity Assay Kit.
Storage/Stability
Upon receipt, the positive control should be removed and stored at -20° or -80°C. The remainder of the kit can be stored for up to 6 months at 2-8°C from the date of shipment. Opened Microplate Wells or reagents may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge.
Note: The kit can be used within one year if the whole kit is stored at -20°C upon receipt. Avoid repeated freeze-thaw cycles.