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Product Description
Specifications
| Size | 1 Plate Kit, 2 Plate Kit, 5 Plate Kit |
| Estimated Lead Time | 1-2 weeks |
| Species | Human |
| Specificity | The olionucleotide/antibody pair provided in this kit recognizes human NF-?B p65 in whole lysates and nuclear extracts. |
| Compatible Sample Types | Cell Lysates, Nuclear Extracts |
| Accession Number | Q04206 |
| Gene Id | 5970 |
| Gene Symbols | RELA, NFKB3 |
| Protein Name / Synonyms | Transcription factor p65 (Nuclear factor NF-kappa-B p65 subunit) (Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3) |
| Quantitative/Semi-Quantitative | Semi-Quantitative |
| Solid Support | 96-well Microplate |
| Design Principle | Sandwich-based |
| Method Of Detection | Colorimetric |
| Research Area | Angiogenesis, Cancer, Inflammation, NFkB Signaling |
| Shipping Type | Dry ice (additional shipping fee) |
| Storage | -20°C |
Introduction
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 p65 from nuclear extracts of HeLa cells or HeLa cells treated with TNF-α. After stimulation with TNF-α, activated NF-κB p65 is translocated into the nucleus where it binds with its corresponding DNA.
Figure 2
Transcription factor activity assay of NF-κB p65 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 p65 to the NF-κB DNA binding site.
Figure 3
Transcription factor activity assay of NF-κB p65 from nuclear extracts of HeLa cells treated with TNF-α and purified NF-κB p50. No cross-reactivity has been observed between NF-κB p65 and p50.
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.
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.
