Mar 30, 2023

Public workspaceGcase co-immunoprecipitation

DOI
https://dx.doi.org/10.17504/protocols.io.14egn2ompg5d/v1
  • michela.deleidi 1,
  • Federico Bertoli1,
  • Maria Jose Perez J.1,
  • Hariam Raji1
  • 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Federico Bertoli
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DOI: https://dx.doi.org/10.17504/protocols.io.14egn2ompg5d/v1
Protocol Citation: michela.deleidi , Federico Bertoli, Maria Jose Perez J., Hariam Raji 2023. Gcase co-immunoprecipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn2ompg5d/v1
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: March 30, 2023
Protocol Integer ID: 79775
Keywords: protein interactions between the enzyme glucocerebrosidase, enzyme glucocerebrosidase, glucocerebrosidase, other proteins in human ipsc, neural precursor cell, protein, derived neural precursor cell, gcase, other protein, protein interaction, immunoprecipitation, enzyme, human ipsc
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.
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Materials

ReagentPierce™ IP Lysis BufferThermo FisherCatalog #87787

ReagentPierce Protease and Phosphatase Inhibitor Mini TabletsThermo FisherCatalog #A32959

ReagentPierce™ Crosslink Magnetic IP/Co-IP KitThermo FisherCatalog #88805

ReagentPierce Protein A/G Magnetic BeadsThermo Fisher ScientificCatalog #88802

Troubleshooting
Gcase co-immunoprecipitation
2h 5m
Wash cells 1X with phosphate-buffered saline (PBS, Sigma‒Aldrich) and detach using Accutase.
Pellet the cell suspension at Centrifigation280 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 Amount25 µL of Pierce protein A/G magnetic beads (Thermo Fisher, #88802-3) twice with 1X Modified Coupling Buffer and incubate with Amount10 µg of GBA MaxPab polyclonal rabbit antibody (Abnova) or normal rabbit IgG (Covalab, #pab01004-P) on a rotating wheel DurationOvernight atTemperature4 °C .
The following day, crosslink the antibody to the beads with a Concentration0.25 millimolar (mM) DSS solution for Duration01:00:00 on a rotating wheel at TemperatureRoom temperature .
1h
Incubate crosslinked magnetic beads DurationOvernight with a total of Amount7 mg of protein for each lysate.
1h
Elute Coimmunoprecipitated proteins from the beads with Amount60 µL of the kit-provided Elution buffer Ph2.0 (Pierce, #88805) and neutralize with Amount6 µL of Neutralization Buffer provided with the kit.
Prepare samples for Western blotting by adding 5x Lane buffer +10% DTT Concentration1 Molarity (M) to a final concentration of 1X.

Note
  • Each western blot input sample loaded corresponds to a total of Amount50 µg of protein.
  • Each western blot CoIP sample loaded corresponded to the total of each elution product (Amount66 µL ).