RTK Array Principle/Protocol

Reagent Preparation

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.*

Array Protocol

Bring all reagents to room temperature before use. Keep samples on ice.

1. Prepare all reagents and samples as directed above.
2. 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.
3. Using flat-tip tweezers, remove each array to be used from between the protective sheets.
4. 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.
5. Incubate for 1 hour on a rocking platform shaker. Orient the tray so that each array rocks end to end in its well.
6. 6. Dilute the lysates to 1.5 mL with Array Buffer 1.
7. Remove Array Buffer 1 from the 4-Well Multi-dish.
8. Add the diluted lysates and place the lid on the 4-Well Multi-dish.
9. Incubate overnight at 2 - 8° C (or 2 hours at room temperature) on a rocking platform shaker.
10. 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.
11. 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.
12. Add 1.5 mL of the freshly diluted Detection Antibody to each well of the 4-Well Multi-dish.
13. Carefully remove each array from its wash container, return it to the 4-Well Multi-dish, and cover with the lid.
14. Incubate for 2 hours at room temperature on a rocking platform shaker.
15. Wash each array as described in steps 10 and 11.
16. 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.
17. Cover with plastic wrap and expose to X-ray film for 1 - 10 minutes.

*Provided this is within the expiration date of the kit.