Immobilized protein a g resin slurry

We used a previously published protocol (43 (link)). Briefly, rat pancreas homogenates (1000 μg of total protein) and amylin-Aβ (30 μM amylin and 16.2 μM Aβ; 1:1) aggregates were incubated with rabbit anti-amylin P2 antibody (1:25, 2 mg/ml stock) and normal rabbit Ig G (#2729; Cell Signaling Technology) as a control, overnight with end-over-end rotation, at 4 °C. Antigen–antibody complex was added to Immobilized Protein A/G resin slurry (20422, Thermo Fisher Scientific) for 2 h at ambient temperature, washed with wash buffer (5 mM of EGTA, 50 mM of Tris, 1% v/v of Triton X-100, pH 7.5 + 1% v/v protease inhibitor, 1% v/v phosphatase inhibitor) and samples eluted with elution buffer (1.5% w/v of glycine, 8% v/v of 1 N HCl, pH 2–3) from the resins using elution buffer. The eluate was used for Western blot analysis.

Proteins were isolated as described above in a modified RIPA buffer containing 0.5% SDS and heated to 95°C for 5 min. Protein lysates were diluted 1:10 in a buffer containing 25 mM Tris and 150 mM NaCl with a pH of 7.2 and incubated with 10 μg of MLK3 antibody overnight at 4°C with constant agitation. The antibody/antigen complex was added to 100 μL of Immobilized Protein A/G resin slurry (Thermo Scientific, Waltham, MA) and incubated with constant agitation at 4°C for 6 hours. Immunoprecipitates were collected by centrifugation according to manufacturer instructions.