Immunoblotting of I3 Neurons and dopaminergic neurons
Pietro De Camilli, Nisha Mohd Rafiq
Abstract
This protocol describes the preparation of cell lysate from and iPSC-derived neurons (I3Neurons, dopaminergic) and the immunoblotting procedure.
Attachments
Steps
Cell culture and treatments
Grow I3 Neurons and dopaminergic (DA) neurons on six-well plates (3-5 × 105 cells/well).
After differentiation in their respective maturation media, wash the neurons with 1mL ice-cold PBS.
Lyse the cells in 200µL 1xRIPA lysis buffer (10X RIPA lysis buffer, Sigma-Aldrich) supplemented with complete™ EDTA-free protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche).
Centrifuge the cells at 13000x g,0h 0m 0s for0h 10m 0s.
Collect the supernatant and store at-20°C.
Determine the protein concentration in sample using Pierce BCA assay (ThermoFisher).
Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
Incubate appropriate volume of cell lysate solution at 95°C for 0h 5m 0s in SDS sample buffer containing 1% 2-mercaptoethanol (Sigma).
Separate the extracted proteins by SDS-PAGE in Mini-PROTEAN TGX precast polyacrylamide gels (Bio-Rad) at 250V and transfer to nitrocellulose membranes (Bio-Rad) at 100 V for 1h 0m 0s or 75 V for 2h 0m 0s (for high molecular weight proteins: >150 kDa).
Block the nitrocellulose membranes for 1h 0m 0s with 5% non-fat milk (AmericanBIO) in TBST (tris-buffered saline [TBS] + 0.1% tween 20), then incubate at 4°C with primary antibodies.
Wash the blots with TBST, thrice, each 0h 5m 0s.
Incubate the blots with IRDye 680RD or 800CW (LI-COR) secondary antibodies (1:8000) for 1h 0m 0s at Room temperature in TBST.
Image blots using the Gel Doc imaging system (Bio-Rad) using manufacturer’s protocols.
