Antigen Information
- P19838
- Human
Assay Format
- B Cell Receptor
- Inflammation
- NFkB Signaling
- T Cell Receptor
- Human
- Cell Lysates
- Nuclear Extracts
- Sandwich-based
- Semi-Quantitative
Product Specifications
Introduction
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
- 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
- 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 < li="">
- Microplate reader capable of measuring absorbance at 450 nm
- 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 activity assay of NF-κB p50 from nuclear extracts of HeLa cells or HeLa cells treated with TNF-α. After stimulation with TNF-α for 30 minutes, activated NF-κB p50 is translocated into the nucleus where it binds with its corresponding DNA. A.Western-blot result of NF-κB p50 from cytoplasm and nuclear fractions. B. Transcription factor activity assay of NF-κB p50 from nuclear fractions with the RayBio®NF-κB p50 Transcription Factor-Activity Assay Kit.
Figure 2 Transcription factor activity assay of NF-κB p50 from nuclear extracts of HeLa cells or HeLa cells treated with TNF-α with the specific competitor or non-specific competitor. The result shows specific binding of NF-κB p50 to the NF-κB canonical DNA binding site.