Pierce™ NFkB p50 Transcription Factor Assay Kit

Catalog number: 89858

Thermo Scientific™  Related applications: Protein Assays and Analysis

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Description

The Thermo Scientific Pierce NFkB p50 Transcription Factor Assay Kit is a complete system containing all the necessary components to measure NF kappa B p50 transcription factor in a 96-well microplate format. Kits contain two streptavidin-coated 96-well plates with the bound biotinylated-consensus sequence for the respective factor. The consensus duplexes bind only the active forms of the target transcription factors, diminishing nonspecific binding. This provides greater signal-to-noise ratios than a traditional ELISA. The captured active transcription factor is then detected with specific primary antibody and a secondary HRP conjugated antibody. Finally, a chemiluminescent substrate is added to the wells, allowing quantitative measurement with a luminometer or CCD camera.

Features of the NFkB p50 Transcription Factor Assay Kit:

Sensitivity—chemiluminescent substrate yields results 20 to 140 times more sensitive than the colorimetric methods with a broader dynamic range
Speed—format allows researcher to perform multiple tests in three to four hours
Reliability—each kit contains a positive control lysate, as well as both wild type and mutant competitor duplexes, ensuring reliable results with a gauge for specificity
High-throughput compatibility—96-well format increases sample throughput over traditional EMSA
Convenience—kits are complete and contain all the reagents for two 96-well assay plates
Versatility—chemiluminescent signal is detectable using a luminometer or CCD Camera

Transcription factors are proteins that bind to promoter regions of genes to regulate their levels of expression. Defects in transcription factor regulation, structure and/or function have been implicated in numerous human diseases such as cancer and inflammation. The ability to screen for transcription factor activation is, therefore, important to drug discovery as well as gene regulation studies.

Traditional methods for measuring active transcription factors include gel-shift or electrophoretic mobility shift assays (EMSA) and colorimetric ELISA-based assays. EMSAs are cumbersome, may involve radioactivity and are not amenable to high-throughput applications. Colorimetric ELISA-based assays have increased speed and throughput, but they have limited sensitivity. Our Transcription Factor Kits are chemiluminescent ELISA-based assays that provide unsurpassed sensitivity and convenience over the existing methodologies.

Additional Figures:

Sensitivity comparison of the Pierce NFk B p50 Kit and other supplier's kits using nuclear extract. Various amounts of TNF-a induced and uninduced HeLa cell nuclear fractions were incubated and detected using the chemiluminescent Pierce NFk B p50 Kit and colorimetric kits from Suppliers A and C. Chemiluminescent results were detected using a luminometer. Colorimetirc results were detected using a plate reader set to the respective wavelengths based on the manufacturer's product instructions. Error bars represent 1 Standard Deviation.
Sensitivity comparison of the Pierce NFk B p50 Kit and other supplier's kits using whole cell extract. Various amounts of TNF-a induced and uninduced HeLa whole cell extracts were incubated and detected using the chemiluminescent Pierce NFk B p65 Kit and colorimetric kits from Suppliers A and C. Chemiluminescent results were detected using a luminometer. Colorimetric results were detected using a plate reader set to the respective wavelengths based on the manufacturer's product instructions. Error bars represent 1 Standard Deviation.

Related Products
Pierce™ NFkB p65 Transcription Factor Assay Kit
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Technique: Enhanced Chemiluminescence, Plate-based Capture
Assay Type: Protein Quantitation Assay
Product Size: 2 x 96 tests
Detection Method: Chemiluminescent
For Use With (Equipment): Luminometer (Microplate)

Contents & storage

• 72 µl Wild-type NFκB Competitor Duplex (-20°C)
• 72 µl Mutant NFκB Competitor Duplex (-20°C)
• 2 × 1.3 ml 5X NFκB Binding Buffer (-20°C)
• 7 µl 2X Poly dI·dC (-20°C)
• 2 µl TNFα Activated HeLa Cell Nuclear Extract Positive Control (-70°C)
• 2 × 96-well NFκB Assay Plate (4°C in foil pouch with desiccant)
• 24 µl NFκB p5 Primary Antibody (4°C)
• 2 µl HRP-conjugated Secondary Antibody (4°C)
• 6 ml Antibody Dilution Buffer (4°C)
• 12 ml Luminol/Enhancer Solution (4°C)
• 12 ml Stable Peroxide Solution (4°C)
• 2 Plate Sealers

Documents

Manuals & protocols