Mar 19, 2024

Public workspaceImmunoblotting of I3 Neurons and dopaminergic neurons

DOI
dx.doi.org/10.17504/protocols.io.3byl49eqjgo5/v1
  • Nisha Mohd Rafiq1,
  • Pietro De Camilli2,3,4,5
  • 1University of Tuebingen;
  • 2Department of Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA.;
  • 3Department of Cell biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.;
  • 4Program in Cellular Neuroscience, Neurodegeneration and Repair.;
  • 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA.
Berrak Ugur
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DOI: dx.doi.org/10.17504/protocols.io.3byl49eqjgo5/v1
Protocol Citation: Nisha Mohd Rafiq, Pietro De Camilli 2024. Immunoblotting of I3 Neurons and dopaminergic neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl49eqjgo5/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 12, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 96917
Keywords: ASAPCRN
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000580
Abstract
This protocol describes the preparation of cell lysate from and iPSC-derived neurons (I3 Neurons, dopaminergic) and the immunoblotting procedure.
Attachments
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17KB
Materials
  • TBS: Concentration50 millimolar (mM) Tris-Cl, Concentration150 millimolar (mM) NaCl, adjust Ph07.5 .
  • TBST: TBS with 0.1% TWEEN-20 (Sigma-Aldrich).

Cell culture and treatments
Cell culture and treatments
10m
10m
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 Amount1 mL ice-cold PBS.
Wash
Lyse the cells in Amount200 µL 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 Centrifigation13000 x g forDuration00:10:00 .
10m
Centrifigation
Collect the supernatant and store atTemperature-20 °C .
Determine the protein concentration in sample using Pierce BCA assay (ThermoFisher).
Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
5h 10m
5h 10m
Incubate appropriate volume of cell lysate solution at Temperature95 °C for Duration00:05:00 in SDS sample buffer containing 1% 2-mercaptoethanol (Sigma).
5m
Incubation
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 Duration01:00:00 or 75 V for Duration02:00:00 (for high molecular weight proteins: >150 kDa).
3h
Block the nitrocellulose membranes for Duration01:00:00 with 5% non-fat milk (AmericanBIO) in TBST (tris-buffered saline [TBS] + 0.1% tween 20), then incubate DurationOvernight at Temperature4 °C with primary antibodies.
Note
Antibody dilutions can be found in Table S1.


1h
Incubation
Overnight
Wash the blots with TBST, thrice, each Duration00:05:00 .

5m
Wash
Incubate the blots with IRDye 680RD or 800CW (LI-COR) secondary antibodies (1:8000) for Duration01:00:00 at TemperatureRoom temperature in TBST.

1h
Incubation
Image blots using the Gel Doc imaging system (Bio-Rad) using manufacturer’s protocols.
Computational step