Pierce protein a g plus agarose beads

MAECs were cross-linked with 250 μM dithiobis(succinimidyl propionate) and lysed with a buffer containing 50 mM Tris HC1 (pH 8), 150 mM NaCl, 1% TX100, 2 mM EDTA and a proteinase inhibitor cocktail. The lysate was incubated sequentially with clean and CAV1 antibody-associated Pierce^™ Protein A/G Plus Agarose beads (ThermoFisher Scientific, Waltham, MA) [19 (link)]. The agarose beads were discarded by centrifugation at 10,000 × g for 1 min. The immunoprecipitants were collected for the determination of proteins.

Sambucus Nigra Agglutinin bound Agarose beads (catalog AL-1303-2, Vector Laboratories) were washed twice with ice-cold PBS (catalog 10010049, Thermo Fisher Scientific). Pierce Protein A/G Plus Agarose beads (catalog 20423, Thermo Fisher Scientific) were used as control. After washing 50 μL of SNA Agarose beads or Protein A/G Plus Agarose beads, they were incubated with 500–1,000 μg cell lysates (collected as described above) in a total of 1 mL volume for 4 hours to overnight in a dark cold room (4°C) on a rotator. Beads were washed twice with ice-cold PBS followed by incubation with Pierce Lane Marker Reducing Sample Buffer (catalog 39000, Thermo Fisher Scientific) for 5 minutes at higher than 90°C for 5 minutes. The pulled lysates were subjected to IB as described above.