Dec 03, 2024
Protein Lysate and Immunoblotting
- Amanda Bentley-DeSousa1,2,3,4,5,
- Shawn Ferguson1,2,3,4,5,6
- 1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 2Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 3Program in Cellular Neuroscience, Neurodegeneration and Repair;
- 4Wu Tsai Institute Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
- 6Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Protocol Citation: Amanda Bentley-DeSousa, Shawn Ferguson 2024. Protein Lysate and Immunoblotting. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo9bmdv4o/v1
Manuscript citation:
https://doi.org/10.1101/2023.10.31.564602
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: December 02, 2024
Last Modified: December 03, 2024
Protocol Integer ID: 113416
Keywords: RAW 264.7, protein lysate, immunoblotting
Funders Acknowledgements:
Aligning Science Across Parkinson’s Disease
Grant ID: ASAP-000580
Abstract
This protocol details steps taken to create protein lysates and perform immunoblotting in RAW 264.7 cells.
Materials
DMEM (Thermo Fisher Scientific, 11965-092)
FBS (Thermo Fisher Scientific, 16140-071)
Penicillin/Streptomycin (Thermo Fisher Scientific, 15140122)
CellStripper (Corning, 356230)
PBS [1.1 mM KH2PO4 (J.T. Baker, 3246-01), 155.2 mM NaCl (Sigma-Aldrich, 3624-05), and 3 mM Na2HPO4 (J.T. Baker, 3828-05)]
Lysis Buffer [50 mM Tris Base (American Bio, AB02000-05000), 150 mM NaCl (Sigma-Aldrich, 3624-05), 1% Triton X-100 (Sigma-Aldrich, X100), 1 mM EDTA (Sigma-Aldrich, 03690) supplemented with cOmplete mini EDTA-free protease inhibitor (Roche, 11836170001) and PhosSTOP (Roche, 4906837001)
Coomassie Plus Protein Assay Reagent (ThermoFisher Scientific, 23236)
Laemmli Buffer [80 mM Tris-HCl pH 6.8 (American Bio, AB020000-05000 / HCl, Sigma-Aldrich, 3624-06), 25.3% Glycerol (American Bio, AB00751), 2.67% SDS (American Bioanalytical, AB01920-00500), and Bromphenol Blue (Sigma-Aldrich, B5525) supplemented with 6.187% fresh b-mercaptoethanol (Sigma-Aldrich, M3148)]
4-15% miniPROTEAN TGX Stain-Free pre-cast gels (BioRad, 4568084/4568085/4568086)
Electrophoresis Buffer [24.76 mM Tris Base (American Bio AB02000-05000), 191.87 mM Glycine (American Bio AB00730-05000), 10 mL 10% SDS (American Bio, AB01920-00500)]
0.45 µm Pore Nitrocellulose Membrane (Thermo Fisher Scientific, 1620115)
Transfer Buffer [24.76 mM Tris Base (American Bio AB02000-05000), 191.87 mM Glycine (American Bio AB00730-05000), 20% Methanol (Sigma-Aldrich, 179337-4l-pB)]
Non-fat dry milk omniblock (American Bio, AB 10109-01000)
TBST [10 mM Tris Base (American Bio AB02000-05000), 150 mM NaCl (Sigma-Aldrich, 3624-05), 0.1% Tween 20 (Sigma-Aldrich, P7949)]
BSA (Sigma-Aldrich, A9647)
SuperSignal West Pico PLUS Chemiluminescence Substrate (Thermo Fisher Scientific, 34580)
SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34095)
Day 0
Day 0
Plate 1 x 10^6 RAW 264.7 cells per well in a 6-well dish.
Day 1
Day 1
1h 41m
1h 41m
Add the indicated compound at the provided concentration and time point in fresh DMEM media containing FBS and P/S.
Proceed directly to the next step if not treating cells.
Wash each dish 2X with cold PBS on ice.
Remove the PBS, scrape each well with 50 µL of ice-cold lysis buffer, and transfer the lysates to Eppendorf tubes.
Lysis Buffer: 50 mM Tris Base, 150 mM NaCl, 1% Triton X-100, and 1 mM EDTA supplemented with cOmplete mini EDTA-free protease inhibitor and PhosSTOP.
Incubate on ice for 00:05:00 .
Centrifuge 14000 rpm, 4°C, 00:08:00 lysates.
8m
Measure the protein concentration of the supernatant using Coomassie Plus Protein Assay Reagent as per the manufacturer’s instructions.
Mix the supernatant/lysate 1:1 with Laemmli Buffer and heat at 95 °C for 00:03:00 .
3m
Laemmli Buffer: 80 mM Tris-HCl pH 6.8, 25.4% Glycerol, 2.67% SDS, and Bromophenol Blue
supplemented with 6.187% fresh B-mercaptoethanol.
Perform electrophoresis using between 20-30 uG of protein in 4-5% miniPROTEAN TGS Stain-Free pre-cast gels.
Electrophoresis Buffer: 24.76 mM Tris Base, 191.87 mM Glycine, and 1% SDS.
After electrophoresis, transfer gels onto 0.45 um pore nitrocellulose membrane at 100V for 01:00:00 .
1h
Transfer Buffer: 24.76 mM Tris Base, 191.87 mM Glycine, and 20% methanol.
Block membranes in 5% non-fat dry milk omniblock in TBST for 00:30:00 .
30m
TBST: 10 mM Tris Base, 150 mM NaCl, 0.1% Tween 20.
Add antibodies in 5% BSA TBST 4 °C Overnight at the indicated concentrations.
Day 2
Day 2
1h 20m
1h 20m
Wash membranes 2X 00:10:00 with TBST.
10m
Add secondary antibodies in either 5% milk TBST (phospho antibodies) or TBST for 01:00:00 at Room temperature .
1h
Wash membranes 3X 00:10:00 with TBST.
10m
Develop membranes via chemiluminescence (SuperSignal West Pico PLUS Chemiluminescence Substrate or SuperSignal West Femto Maximum Sensitivity Substrate) and image with a Biorad Chemidoc MP Imaging Station.