License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 30, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 79780
Keywords: ASAPCRN
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.
Pierce™ IP Lysis BufferThermo FisherCatalog #87787
Pierce Protease and Phosphatase Inhibitor Mini TabletsThermo FisherCatalog #A32959
Pierce™ Crosslink Magnetic IP/Co-IP KitThermo FisherCatalog #88805
Pierce Protein A/G Magnetic BeadsThermo Fisher ScientificCatalog #88802
Gcase co-immunoprecipitation
Gcase co-immunoprecipitation
2h 5m
2h 5m
Wash cells 1X with phosphate-buffered saline (PBS, Sigma‒Aldrich) and detach using Accutase.
Pellet the cell suspension at 280 rcf, 23°C, 00:05:00.
5m
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 25 µL of Pierce protein A/G magnetic beads (Thermo Fisher, #88802-3) twice with 1X Modified Coupling Buffer and incubate with 10 µg of GBA MaxPab polyclonal rabbit antibody (Abnova) or normal rabbit IgG (Covalab, #pab01004-P) on a rotating wheel Overnight at4 °C.
The following day, crosslink the antibody to the beads with a 0.25 millimolar (mM) DSS solution for 01:00:00 on a rotating wheel at Room temperature.
1h
Incubate crosslinked magnetic beads Overnight with a total of 7 mg of protein for each lysate.
1h
Elute Coimmunoprecipitated proteins from the beads with 60 µL of the kit-provided Elution buffer 2.0 (Pierce, #88805) and neutralize with 6 µL of Neutralization Buffer provided with the kit.
Prepare samples for Western blotting by adding 5x Lane buffer +10% DTT 1 Molarity (M) to a final concentration of 1X.
Note
Each western blot input sample loaded corresponds to a total of 50 µg of protein.
Each western blot CoIP sample loaded corresponded to the total of each elution product (66 µL).