Proteome Profiler Human NFkB Pathway Array

A membrane-based antibody array for the parallel determination of the relative levels of selected human NFkB Pathway proteins. Validated for analyte detection in cell lysates.

Principle of the Assay

Carefully selected capture antibodies have been spotted in duplicate on nitrocellulose membranes. Cell lysates are diluted and mixed with a cocktail of biotinylated detection antibodies. The sample/antibody mixture is then incubated with the array. Any protein/detection antibody complex present is bound by its cognate immobilized capture antibody on the membrane. Streptavidin-Horseradish Peroxidase and chemiluminescent detection reagents are added, and a signal is produced in proportion to the amount of protein bound. Chemiluminescence is detected in the same manner as a Western blot.

Why Use an Antibody Array to Detect Multiple Proteins?

Determining the expression of multiple Proteins in a single sample can be expensive, time consuming and can require specialized equipment. Performing multiple immunoprecipitations and Western blots requires time, labor, and reagents. The use of a multiplex antibody array to detect multiple proteins in a single sample can be cost-effective and also save time and sample.

• 4 Array Membranes
• 4-Well Multi-dish
• Array Buffers
• Wash Buffer
• Detection Antibody Cocktail, Human NF kappa B Pathway Array
• Streptavidin-HRP
• Chemiluminescent Detection Reagents
• Transparency Overlay Template
• Detailed Protocol

Stability and Storage

Reagents are stable for 12 months from date of receipt when stored in the dark at 2^° C to 8^° C.
 

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HCT-116 Untreated vs. Epoxomicin Treated HCT-116 human colorectal carcinoma cells were untreated or treated with 1 µM Epoxomicin (Boston Biochem, Catalog # I-110) for 6 hours (500 µg lysate, 5 minute exposure). RS = Reference Spots.

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Daudi Untreated vs. IFN-alpha 2A Treated Daudi human Burkitt’s lymphoma cells were untreated or treated with 500 U/mL IFN-alpha 2A (R&D Systems, Catalog # 11100-1) for five minutes (1 mg lysate, 10 minute exposure).

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HT-29 Untreated vs. TNF-alpha +/- MLN4924 Treatment HT-29 human colorectal carcinoma cells were untreated or treated with 50 ng/mL recombinant human TNF-alpha (R&D Systems, Catalog # 210-TA) for ten minutes with or without pretreatment with 5 µM MLN4924 (Boston Biochem, Catalog # I-502) for 1 hour (400 µg lysate, 5 minute exposure).