Proteome Profiler Mouse XL Cytokine Array

Assay Procedure

Refer to the product datasheet for complete product details.

View Graphic Procedure

• 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)

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

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.

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

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