Gcase co-immunoprecipitation
michela.deleidi, Federico Bertoli
Abstract
We developed this protocol to identify protein-protein interactions between the enzyme glucocerebrosidase (GCase) and other proteins in human iPSC-derived Neural Precursor Cells.
Attachments
676- 1426.docx
Steps
Gcase co-immunoprecipitation
Wash cells 1X with phosphate-buffered saline (PBS, Sigma‒Aldrich) and detach using Accutase.
Pellet the cell suspension at 280rcf,23°C.
Lyse the pellets in IP/lysis buffer (Thermo Fisher, #87787) supplemented with a protease/phosphatase inhibitor cocktail (Pierce, #A32959).
Carry out coimmunoprecipitation using the Thermo Fisher Pierce Crosslink Magnetic IP/Co-IP Kit (#88805) according to the manufacturer´s instructions:
Prewash 25µL of Pierce protein A/G magnetic beads (Thermo Fisher, #88802-3) twice with 1X Modified Coupling Buffer and incubate with 10µg of GBA MaxPab polyclonal rabbit antibody (Abnova) or normal rabbit IgG (Covalab, #pab01004-P) on a rotating wheel at4°C.
The following day, crosslink the antibody to the beads with a 0.25millimolar (mM) DSS solution for 1h 0m 0s on a rotating wheel at Room temperature.
Incubate crosslinked magnetic beads 1h 0m 0s with a total of 7mg of protein for each lysate.
Elute Coimmunoprecipitated proteins from the beads with 60µL of the kit-provided Elution buffer 2.0 (Pierce, #88805) and neutralize with 6µL of Neutralization Buffer provided with the kit.
Prepare samples for Western blotting by adding 5x Lane buffer +10% DTT 1Molarity (M) to a final concentration of 1X.
