Alternative Biotinylation Protocol for the Phospho-Immunoreceptor Array

Introduction

Biotinylation is a commonly used method for labeling of cell-surface proteins. This method can be combined with antibody array analysis for profiling cell surface-expressed proteins. In this protocol the user prepares samples by labeling cell-surface proteins with Sulfo-NHS-LC-Biotin and lysing cells in a non-denaturing lysis buffer. For array analysis, cell lysates are diluted and incubated with the Human Phospho-Immunoreceptor Array (Catalog # ARY004B). After binding of immunoreceptors to their specific capture antibodies on the array, unbound material is washed away. Streptavidin-HRP and chemiluminescent detection is then used to visualize biotinylated immunoreceptors corresponding to those expressed on the cell surface.

Sample Biotinylation Data

Required Reagents

• PBS (adjusted to pH 8.0)
• Sulfo-NHS-LC-Biotin (Pierce, Catalog # 21335)
• Glycine
• Streptavidin-HRP (Catalog # DY998)
• Chemiluminescence Detection Reagents

Cell-surface biotinylation and Sample Preparation

The cell-surface biotinylation procedure is a modification of the procedure recommended by the manufacturer of the biotinylation reagent. For suspended cells, wash two times with ice-cold PBS (pH 8.0) to remove sources of primary amines in the culture media. After the final spin, resuspend the cells at a concentration of 2.5 x 10⁷ cells/mL in ice-cold PBS (pH 8.0) containing 0.25 mg of Sulfo-NHS-LC-Biotin reagent per mL and incubate at 4 °C for 30 minutes. Wash the cells twice with PBS containing 100 mM glycine to quench and remove excess biotin reagent. Solubilize the cells at 1 x 10⁷ cells/mL in Lysis Buffer 15. Pipette up and down to resuspend and rock the lysates gently at 2 to 8 °C for 30 minutes. Microcentrifuge at 14,000 x g for 5 minutes, and transfer the supernatant into a clean microcentrifuge tube. Determine the sample protein concentration using a total protein assay. For incubation with the Human Phospho-Immunoreceptor Array, use a smaller quantity of lysate than that used for immunoprecipitation or Western blot (5 to 50 µg ). Lysates should be used immediately or aliquoted and stored at ≤ 70 °C. Thawed lysates should be kept on ice prior to use.

Array Protocol

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

1. Add 1.5 mL of Array Buffer 1 into individual wells of the 4-Well Multi-dish that will be used for each array.
2. Using flat-tip tweezers, remove each array to be used from between the protective sheets.
3. 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.
4. Incubate for 1 hour on a rocking platform shaker. Orient the tray so that each array rocks end to end in its well.
5. Dilute the lysate to 1.5 mL with Array Buffer 1 and add dilute lysate.
6. Remove Array Buffer 1 from the 4-Well Multi-dish.
7. Incubate overnight at 2 to 8 º C (or 2 hours at room temperature) on a rocking platform shaker. Orient the tray so that each array rocks end to end in its well.
8. 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.
9. Wash each array with 1X Wash Buffer for 10 minutes on a rocking platform shaker. Repeat two times for a total of three washes.
10. Dilute Streptavidin-HRP 1:4000 by adding 2.5 µL of Streptavidin-HRP to 10 mL of Array Buffer 2.
11. Add 1.5 mL of the freshly diluted Streptavidin-HRP to each well of the 4-Well Multi-dish.
12. Carefully remove each array from its wash container, return it to the 4-Well Multi-dish, and cover with the lid.
13. Incubate for 1 hour at room temperature on a rocking platform shaker. Orient the tray so that each array rocks end to end in its well.
14. Wash each array as described in steps 8 and 9.
15. Remove each array from the wash container and place it on a plastic sheet protector. Expose each array to chemiluminescent substrate according to instructions provided in the kit insert.
16. Cover with plastic wrap and expose to X-ray film for 1 to 10 minutes.

Sample Data

Cell-surface Biotinylation Profiles of Immunoreceptors in Selected Cell Lines

Lysates from biotinylated NK-92 cells (A; 25 µg), biotinylated THP-1 cells (B ; 20 µg), and unbiotinylated THP-1 cells (C;25 µg) were tested with the Human Phospho-Immunoreceptor Array using the above protocol. The results reveal the immunoreceptor expression profile of each cell line and only positive control spots in unbiotinylated cells.