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Antibody Array Assay Protocol for Protein Extraction and Detection

Learn how to extract proteins, label them with biotin, and detect them using an antibody microarray assay. Follow detailed steps for a successful experiment.

tavarius-josiah
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Antibody Array Assay Protocol for Protein Extraction and Detection

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  1. Antibody Array Assay Report

  2. Protocol • Protein Extraction • Wash the cells with ice cold 1X PBS. • Add Lysis Beads and Extraction Buffer to the sample. • Mix rigorously by vortexing for 30 seconds. Incubate the mixture on ice for 10 minutes. • Repeat vortexing for 30 seconds at 10-minute intervals for 60 minutes. Incubate the mixture on ice between vortexing. • Centrifuge the mixture at 10,000 x g for 20 minutes at 4°C. Transfer the supernatant to a clean tube. • Use Pro-Spin columns to change the buffer in the supernatant to Labeling Buffer. • Measure protein concentration. • Protein Labeling • Add 100 uL of DMF to 1 mg of Biotin Reagent to give a final concentration of 10 ug/uL. • Add Labeling Buffer to the protein sample to bring the volume to 75 uL. • Add 3 uL of the Biotin/DMF to the protein sample with Labeling Buffer. • Mix and incubate at room temperature for two hours with mixing. • Add 35 uL of Stop Reagent. Incubate for 30 minutes at room temperature with mixing. • Coupling • Blocking: Submerge Antibody Microarray in Blocking Buffer. Shake for 40 minutes at room temperature. • Rinse the slide with Milli-Q grade water. • Incubate the slide in Coupling Chamber with 100 ug of labeled protein sample in 6 mL Coupling Solution on an orbital shaker for 2 hours at room temperature. • Remove the slide from the coupling chamber. • Wash the slide three times with fresh Wash Buffer. • Rinse extensively with DI water. • Detection • Add 30 ul of Cy3-Streptavidin (1 mg/ml) to the 60-ml bottle containing Detection Buffer. • Submerge the slide in 30 ml of Cy3-Streptavidin solution. • Incubate on an orbital shaker for 45 minutes at room temperature in the dark. • Wash the slide three times with fresh Wash Buffer. • Rinse extensively with DI water. • Dry the slide with compressed nitrogen. • Scan on Axon GenePix Array Scanner.

  3. Assay Results Phospho Explorer Antibody Array Data (Excel file) Assay Images Page 4

  4. Array Images Phospho Explorer Antibody Array Control Sample SAR Sample

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