Bring all reagents to room temperature before use.
Human Phospho-RTK Array - Four nitrocellulose membranes each containing 42 different anti-RTK antibodies and 6 controls printed in duplicate. Handle arrays only with gloved hands and flat-tipped tweezers. After opening, reseal unused membranes in the foil pouch with desiccant and store at 2 - 8° C for up to 3 months.*
Anti-Phospho-Tyrosine-HRP Detection Antibody - One vial of 700 µg/mL mouse anti-phospho-tyrosine antibody conjugated to HRP. Immediately before each use, dilute the Detection Antibody in 1X Array Buffer 2 to a working concentration of 70 ng/mL. Store at
2 - 8° C for up to 3 months after initial use. * DO NOT FREEZE.
Array Buffer 1 - Ready for use. Store at 2 - 8° C for up to 3 months after initial use.*
1X Array Buffer 2 - Dilute 2 mL of Array Buffer 2 Concentrate into 8 mL of deionized or distilled water. Prepare fresh for each use.
1X Wash Buffer - Dilute 20 mL of Wash Buffer Concentrate into 480 mL of deionized or distilled water. Store at 2 - 8° C for up to 3 months after initial use.*
Bring all reagents to room temperature before use. Keep samples on ice.
- Prepare all reagents and samples as directed above.
- For blocking each array, add 1.5 mL of Array Buffer 1 to each well of the 4-Well Multi-dish which will be used.
- Using flat-tip tweezers, remove each array to be used from between the protective sheets.
- Place one array into each well of the 4-Well Multi-dish. The array number should be facing upward.
Note: The blue dye will disappear from the spots. The capture antibodies are retained in their specific locations.
- Incubate for 1 hour on a rocking platform shaker. Orient the tray so that each array rocks end to end in its well.
- 6. Dilute the lysates to 1.5 mL with Array Buffer 1.
- Remove Array Buffer 1 from the 4-Well Multi-dish.
- Add the diluted lysates and place the lid on the 4-Well Multi-dish.
- Incubate overnight at 2 - 8° C (or 2 hours at room temperature) on a rocking platform shaker.
- Carefully remove each array and place into individual plastic containers with a minimum of 20 mL of 1X Wash Buffer. Rinse the 4-Well Multi-dish with deionized or distilled water and dry thoroughly.
- Wash each array with 1X Wash Buffer by soaking for 10 minutes on a rocking platform shaker. Repeat two times for a total of three washes.
- Add 1.5 mL of the freshly diluted Detection Antibody to each well of the 4-Well Multi-dish.
- Carefully remove each array from its wash container, return it to the 4-Well Multi-dish, and cover with the lid.
- Incubate for 2 hours at room temperature on a rocking platform shaker.
- Wash each array as described in steps 10 and 11.
- Remove each array from the wash container and place it on a plastic sheet protector. Expose each array to chemiluminescent reagents such as R&D Systems Western Glo (Catalog # ARY004B) or equivalent.
- Cover with plastic wrap and expose to X-ray film for 1 - 10 minutes.
*Provided this is within the expiration date of the kit.