NF-κB is a transcription factor that plays a pivotal role in a number of physiological cell processes such as growth, apoptosis, and immune response through regulating the expression of a number of cellular genes. The NF-κB family includes five members: cRel, RelB, p65(RelA), p50(p105, precursor of p50), and p52(p100, precursor of p52), which form homo- or heterodimers when in active status. Normally, NF-κB is inactive and retained in the cytoplasm bound to its inhibitory protein IκB. Upon stimulation of various factors such as infection of pathogens including viruses and bacteria or treatment of cytokines such as IL-1 or TNF-α, IκB proteins are phosphorylated, ubiquitinated and finally degraded. Released NF-κB proteins then form homo- or heterodimers, most commonly p50/p65 or p50/p50, which are transported into the nucleus where they bind to specific DNA sequences of targeted genes and activate transcription. NF-κB also induces expression of its own inhibitor, IκBa, which binds to active NF-κB in the nucleus to terminate the induction of gene expression.
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 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.
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.
