The RayBio® ATF-1 Transcription Factor Activity Assay Kit uses a dsDNA coated plate with canonical ATF-1 binding sequences to semi-quantitatively detect active ATF-1 in lysates or nuclear extracts. Dry ice shipment (additional fee).
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.
