Proteome Profiler Mouse XL Cytokine Array Best Seller
R&D Systems, part of Bio-Techne | Catalog # ARY028
Key Product Details
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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). |
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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). |
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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
- 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.
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.
