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About 2 mg of cell lysate in 1 mL RIPA buffer was incubated with 2 µg of anti-EGFR antibody with shaking at 4°C overnight. Samples were then mixed with 30 µL of protein A/G-agarose (Santa Cruz) and incubated with shaking at 4°C for 2 hours. Agarose beads were washed five times in RIPA buffer. Immunoprecipitates were then eluted in 2 X SDS sample loading buffer and separated by SDS-PAGE.
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