Dec 16, 2024
Western Blotting for Neuronal Proteins
- Mukesh Kumar1,
- Timothy A. Ryan1,2
- 1Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, Maryland 20815, USA
Protocol Citation: Mukesh Kumar, Timothy A. Ryan 2024. Western Blotting for Neuronal Proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wqdjvo5/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: November 26, 2024
Last Modified: December 16, 2024
Protocol Integer ID: 114389
Keywords: ASAPCRN
Funders Acknowledgements:
NIH
Grant ID: NS036942
NIH
Grant ID: NS11739
ASAP
Grant ID: ASAP-024404
Abstract
This protocol outlines a detailed procedure for neuronal protein extraction and western blot analysis, enabling precise detection and quantification of target proteins.
Guidelines
The protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Materials
- Neurons cultured from neonatal P0-1 rodent brain
- Poly-D-lysine: (SigmaAldrich, P7886)
- Lentivirus (produced as per lentivirus production protocol)
- RIPA lysis buffer (common reagents procured from Sigma):
| A | B | |
| Tris-HCl, pH 7.6 | 25 mM | |
| NaCl | 150 mM | |
| NP-40 | 1% | |
| sodium deoxycholate | 1% | |
| SDS | 0.1% | |
| Complete protease inhibitor | (Roche, 11697498001) |
- cOmplete™ Protease Inhibitor CocktailMerck MilliporeSigma (Sigma-Aldrich)Catalog #11697498001
- Pierce BCA Protein Assay Kit Thermo Fisher ScientificCatalog #23225
- 4x Laemmli Sample BufferBio-Rad LaboratoriesCatalog #1610747
- Immobilon®-FL PVDF MembraneMerck MilliporeSigma (Sigma-Aldrich)Catalog #Ipfl00010
- Li-Cor Intercept (TBS) blocking buffer (Li-Cor, P/N: 927-60001)LI-CORCatalog #P/N: 927-60001
- Anti-CPT2 antibody [EPR13626] - C-terminalAbcamCatalog #ab181114
- GAPDH (D16H11) XP® Rabbit mAb #5174Cell Signaling TechnologyCatalog #5174S
- HRP-conjugated secondary antibodies:
- Invitrogen, 31430 (anti-mouse),
- Invitrogen, 31460 (anti-rabbit)
- Immobilon Forte Western HRP substrate, 100 mLMerck MilliporeSigma (Sigma-Aldrich)Catalog #WBLUF0100
Equipments:
- Tissue culture incubator
- Centrifuge
- BioRad SDS-PAGE apparatus
- BioRad Western blot transfer system
- LiCor Odyssey Fc system.
Neuronal Culture and Lentivirus Infection
Neuronal Culture and Lentivirus Infection
3d
3d
Isolate neurons from rat pups (P0-1 days) and plate them on 35 mm dishes coated with 50 µL poly-D-lysine.
Culture the neurons under standard conditions (37 °C , 5% CO₂) and allow them to adhere.
At 2 days in vitro (DIV-2), introduce lentivirus into the culture media at an appropriate multiplicity of infection (MOI) tested previously for individual virus prepared.
Incubate the neurons with lentivirus for 48:00:00 -72:00:00 to ensure efficient transduction.
3d
Protein Extraction
Protein Extraction
55m
55m
Remove the culture media and rinse neurons gently with ice-cold PBS.
Add RIPA lysis buffer (supplemented with complete protease inhibitor) to the dish.
Scrape the neurons into the lysis buffer using a cell scraper.
Transfer the lysate to a microcentrifuge tube and incubate On ice for 00:20:00 .
40m
Centrifuge the lysate at 14000 rpm, 4°C, 00:15:00 to remove debris.
15m
Transfer the supernatant (cytosolic protein fraction) to a new tube.
Protein Quantification
Protein Quantification
Measure protein concentration using the BCA Protein Assay Kit according to the manufacturer’s instructions.
Normalize the protein concentration of all samples.
SDS-PAGE and Transfer to PVDF membrane
SDS-PAGE and Transfer to PVDF membrane
5m
5m
Mix equal amounts of protein with SDS sample buffer (2X) and heat at 95 °C for 00:05:00 to denature the proteins.
5m
Load the samples onto an SDS-PAGE gel and run electrophoresis at the appropriate voltage.
Transfer the separated proteins onto a PVDF membrane using a wet transfer system.
Membrane Blocking
Membrane Blocking
1h
1h
Block the PVDF membrane in LiCor blocking buffer for 01:00:00 at Room temperature with gentle agitation.
1h
Primary Antibody Incubation
Primary Antibody Incubation
1h
1h
Dilute the primary antibodies in LiCor blocking buffer according to the manufacturer’s instructions.
Incubate the membrane with the primary antibody Overnight at 4 °C with gentle rocking.
1h
Secondary Antibody Incubation
Secondary Antibody Incubation
1h 30m
1h 30m
Wash the membranes 3 times with TBST (Tris-buffered saline with 0.1% Tween-20), 10 minutes per wash.
Wash the membranes with TBST (Tris-buffered saline with 0.1% Tween-20) for 00:10:00 (1/3).
10m
Wash the membranes with TBST (Tris-buffered saline with 0.1% Tween-20) for 00:10:00 (2/3).
10m
Wash the membranes with TBST (Tris-buffered saline with 0.1% Tween-20) for 00:10:00 (3/3).
10m
Dilute HRP-conjugated secondary antibody in LiCor blocking buffer and incubate the membrane for 01:00:00 at Room temperature .
1h
Signal Detection
Signal Detection
30m
30m
Wash the membrane 3 times with TBST, 10 minutes per wash.
Wash the membrane with TBST for 00:10:00 (1/3).
10m
Wash the membrane with TBST for 00:10:00 (2/3).
10m
Wash the membrane with TBST for 00:10:00 (3/3).
10m
Apply ECL reagent to the PVDF membrane.
Capture chemiluminescent signals using the LiCor Odyssey Fc system.
Note
• Ensure all steps are performed on ice during protein extraction to prevent protein degradation.
• Optimize antibody dilutions and incubation times for specific targets.
• Blotting-Grade Blocker (Bio-Rad, 1706404) can be used in place of LiCor blocking buffer.
• Handle all reagents and equipment following standard laboratory safety protocols.