Proteome Profiler Mouse XL Cytokine Array Best Seller

A membrane-based antibody array for the parallel determination of the relative levels of selected mouse cytokines and chemokines. Validated for analyte detection in cell culture supernates, cell lysates, tissue lysates, serum, and urine.

Principle of the Assay

The Proteome Profiler Mouse XL Cytokine Array Kit is a membrane-based sandwich immunoassay. Capture antibodies spotted in duplicate on nitrocellulose membranes bind to specific target proteins present in the sample (Step 1). Captured proteins are detected with biotinylated detection antibodies (Step 2) and then visualized using chemiluminescent detection reagents (Step 3). The signal produced is proportional to the amount of analyte bound.

Why Use an Antibody Array to Detect Multiple Cytokines?

Determining the expression of multiple cytokines 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 cytokines in a single sample can be cost-effective and also save time and sample.

• Rectangular 4-Well Multi-dish
• 4 Mouse XL Cytokine Array nitrocellulose membranes spotted with 111 different antibodies to mouse cytokines
• Array Buffer 4
• Array Buffer 6
• Chemi Reagent 1
• Chemi Reagent 2
• Detection Antibody Cocktail, Mouse XL Cytokine Array
• Streptavidin-HRP
• Transparency Overlay Template
• Wash Buffer Concentrate (25X)

For a complete list of the kit contents and necessary materials, please see the Materials Provided/Other Supplies Required sections of the product datasheet.

Assays for Analytes represented in the Mouse XL Cytokine Array Kit

Scientific Data Examples for Proteome Profiler Mouse XL Cytokine Array

	Detection of Selected Mouse Cytokines in Balb/3T3 Cell Line Lysate. Lysates from Balb/3T3 mouse embryonic fibroblast cells were untreated or treated with 100 ng/mL of recombinant mouse TNF-alpha (R&D Systems, Catalog # 410-MT) for 24 hours (200 µg lysate, 10 minute exposure).
	Detection of Selected Mouse Cytokines in CMT-93 Cell Line Lysate. Lysates from CMT-93 mouse rectal carcinoma cells were untreated or treated with 100 ng/mL LPS for 24 hours (200 µg lysate, 10 minute exposure).
	Detection of Selected Mouse Cytokines in Balb/3T3 Cell Line Supernate. Balb/3T3 mouse embryonic fibroblast cells were untreated or treated with 100 ng/mL of recombinant mouse TNF-alpha (R&D Systems, Catalog # 410-MT) for 24 hours (500 µL of cell culture supernate, 5 minute exposure).

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, relative expression levels of mouse cytokines and chemokines in samples can be determined using the following procedure:

• Prepare membrane and incubate with prepared sample
• Incubate the membrane with Detection Antibody Cocktail
• Incubate the membrane array with Streptavidin-HRP
• Develop the membrane array with Chemi Reagents 1 and 2
• Expose the membrane array to autoradiography film

View Graphic Procedure

 

 

Kit Contents

• Rectangular 4-Well Multi-dish
• 4 Mouse XL Cytokine Array nitrocellulose membranes spotted with 111 different antibodies to mouse cytokines
• Array Buffer 4
• Array Buffer 6
• Chemi Reagent 1
• Chemi Reagent 2
• Detection Antibody Cocktail, Mouse XL Cytokine Array
• Streptavidin-HRP
• Transparency Overlay Template
• Wash Buffer Concentrate (25X)

 

Other Supplies Required

Reagents

• Aprotinin
• Leupeptin (Catalog #EI002)
• Pepstatin (Catalog #EI003)
• Igepal^® CA-630
• Deionized or distilled water

Materials

• Pipettes and pipette tips
• Gloves
• Plastic container with the capacity to hold 50 mL (for washing the arrays)
• Plastic transparent sheet protector (trimmed to 10 cm x 12 cm and open on three sides)
• Plastic wrap
• Absorbent lab wipes (KimWipes^® or equivalent)
• Paper towels
• X-ray film (Kodak BioMax™ Light-1) or equivalent
• Flat-tipped tweezers

Equipment

• Rocking platform shaker
• Microcentrifuge
• Autoradiography cassette
• Film developer
• Flatbed scanner with transparency adapter capable of transmission mode
• Computer capable of running image analysis software and Microsoft Excel

Other Supplies Required for Cell Lysate Samples

• Phosphate-Buffered Saline (PBS)
• Lysis buffer (1% Igepal CA-630, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 10 µg/mL Aprotinin, 10 µg/mL Leupeptin, and 10 µg/mL Pepstatin)

Other Supplies Required for Tissue Lysate Samples

• PBS with protease inhibitors (10 µg/mL Aprotinin, 10 µg/mL Leupeptin, and 10 µg/mL Pepstatin)
• Triton^™ X-100

 

Procedure Overview

R&D Systems Protocol for Multiple Analyte Detection Using the Proteome Profiler^™ Mouse XL Cytokine Array Kit, Panel A (Catalog # ARY028)

Add 2 mL of Array Buffer 6 to each well of the supplied 4-Well Multi-dish.

Place each array membrane in a separate well of the 4-Well Multi-dish.

Incubate for one hour on a rocking platform shaker.

Add 0.5 mL Array Buffer 4 to each sample.

Adjust volume of each sample to final volume of 1.5 mL with Array Buffer 6.

Replace the Array Buffer 6 in each well of the 4-Well Multi-dish with prepared samples.

Incubate overnight at 2 °C to 8 °C on a rocking platform.

Wash each array membrane 3 times with 1X Wash Buffer in a separate container.

Wash each well of the 4-Well Multi-dish with 1X Wash Buffer.

Add 30 µL of Detection Antibody Cocktail to 1.5 mL of Array Buffer 4/6 for each array.

Pipette 1.5 mL diluted Detection Antibody Cocktail into each well of the 4-Well Multi-dish.

Return each array membrane to the 4-Well Multi-dish containing diluted Detection Antibody Cocktail.

Incubate for one hour on a rocking platform shaker.

Wash each array membrane 3 times with 1X Wash Buffer in a separate container.

Wash each well of the 4-Well Multi-dish with 1X Wash Buffer.

Add 2 mL of diluted Streptavidin-HRP to each well of the 4-Well Multi-dish.

Place the array membrane in the diluted Streptavidin-HRP solution.

Incubate for 30 minutes on a rocking platform shaker.

Wash each array membrane 3 times with 1X Wash Buffer in a separate container.

Place the array membrane on a plastic sheet protector.

Pipette 1 mL of the prepared Chemi Reagent Mix evenly onto the membrane.

Cover the membrane with the top sheet of the plastic protector.

Incubate for 1 minute.

Blot off excess Chemi Reagent Mix.

Wrap the membrane and sheet protector in plastic wrap.

Place the wrapped array membrane in an autoradiography film cassette and expose to X-ray film.