Oct 21, 2025
Immunoblotting
Forked from Immunoblotting
- Devin M. Fuller1,2,
- Thomas Melia1,2
- 1Department of Cell Biology, Yale University School of Medicine, New Haven, CT;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, 20 MD
Protocol Citation: Devin M. Fuller, Thomas Melia 2025. Immunoblotting. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmyxyov3p/v1
Manuscript citation:
Fuller Devin M, Wu Yumei, Schueder Florian, Rasool Burha, Nag Shanta, Korfhage Justin L, Garcia-Milian Rolando, Melnyk Katerina D, Bewersdorf Joerg, De Camilli Pietro, Melia Thomas J (2025) ATG2A engages RAB1A and ARFGAP1 positive membranes during autophagosome biogenesis eLife 14:RP107316
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 31, 2025
Last Modified: October 21, 2025
Protocol Integer ID: 125784
Keywords: Immunoblot, Western blot, Tris-glycine, Tris-acetate, siRNA, cGAMP, HT-DNA, ASAPCRN, cultured cell, collection of protein, protein, protocol
Funders Acknowledgements:
National Institutes of Health
Grant ID: R01 GM100930
National Institutes of Health
Grant ID: R35 GM153482
National Institutes of Health
Grant ID: F31 AG079606
Aligning Science Across Parkinson’s
Grant ID: ASAP-025173
Abstract
This protocol describes collection of protein from cultured cells and immunoblotting
Materials
Cell culture materials:
DMEM (Thermo Fisher Scientific, 11965-092)
FBS (Thermo Fisher Scientific, 16140-071)
Penicillin/Streptomicin (10,000 U/mL; Thermo Fisher Scientific, 15140122)
PBS (Thermo Fisher Scientific, 10010023)
Earle’s Balanced Salt Solution (EBSS; Thermo Fisher Scientific, 24010043)
Transfection Reagents:
Opti-Mem (Thermo Fisher Scientific, 31985062)
Lipofectamine 3000 (Thermo Fisher Scientific, L3000008)
Dharmafect (Horizon Discovery, T-2001-01)
Lysis Buffer:
lysis buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) with Protease Inhibitor Cocktail
Tris (American Bio, AB02000-05000)
NaCl (Sigma-Aldrich, S9888-25G)
EDTA (Sigma-Aldrich, 3690)
Nonidet P40 Substitute (Roche, 11754599001)
COmplete mini EDTA Free (Roche, 11836170001)
Troubleshooting
Cell culture and treatments
2d
Culture the HEK293 cells at 37 °C in 5% CO2 and DMEM containing 10% FBS, 1000 U/mL penicillin, a 1 mg/mL streptomycin.
For any given experiment, plate the cells at such density so as to be approximately 90% confluent at the time of lysis.
For experiments using siRNA, transfect a final concentration of 25 nM of the indicated siRNA using 4 µL Dharmafect (Horizon Discovery) in Opti-MEM (Gibco) per well of a six well dish according to manufacturer protocol. Lyse the cells 48:00:00 after siRNA transfection.
2d
Cell lysis and sample preparation
18m
Supplement lysis buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) with Protease Inhibitor Cocktail (Roche) and chill On ice .
Aspirate media from cells, add PBS, and scrape cells using a cell scraper (Corning) On ice
Pipette the resuspended cells into Eppendorf tube.
Centrifuge at 500 x g, 4°C, 00:03:00 , aspirate the excess PBS.
3m
Pipette 150 uL lysis buffer onto cell pellet, pipette mixture up and down 10 times with a P-200 pipette tip
Note
NOTE: Take care not to introduce bubbles.
Incubate Eppendorf tube On ice for 00:05:00 .
5m
Determine protein concentration in sample using Bradford Assay.
Prepare samples at desired concentration, add 1 mM DTT and 1x LDS loading buffer.
Gel electrophoresis and immunoblotting
5h 20m
Incubate samples at 95 °C for 00:05:00 .
5m
During this incubation, prepare gel apparatus with NuPAGE‱ Bis-Tris Mini Protein Gels, 4–12%, 1.0–1.5 mm (ThermoFisher) and 1x MOPS running buffer.
Load samples into gel and run until dye front reaches bottom (180 V, roughly 60 min).
Remove gel and set up transfer cassette with nitrocellulose membrane.
Transfer at 30-35 V for 01:30:00 in 1x transfer buffer.
1h 30m
Block membrane with 5% BSA in PBST for 01:00:00 at 22 °C .
1h
Incubate membrane with primary antibodies in 5% BSA and 0.03% sodium
azide in PBST Overnight at 4 °C .
Note
NOTE: Optimal primary antibody incubation time and temperature can be determined empirically for a given primary antibody
1h
Wash membrane with PBST for five minutes. Repeat a total of 3 times.
Wash membrane for 00:05:00 with PBST (1/3).
5m
Wash membrane for 00:05:00 with PBST (2/3).
5m
Wash membrane for 00:05:00 with PBST (3/3).
5m
Incubate membrane with secondary antibodies (1:5,000) in 5% milk in PBST for 01:00:00 at 22 °C .
1h
Wash membrane with PBST. Repeat a total of 3 times.
Wash membrane for 00:05:00 with PBST (1/3).
5m
Wash membrane for 00:05:00 with PBST (2/3).
5m
Wash membrane for 00:05:00 with PBST (3/3).
5m
Image membranes using a BioRad VersaDoc imaging system.
Acknowledgements
This work was supported by grants from the National Institutes of Health (R01 GM100930 and R35 GM153482 to TJM; R01 GM151829 to JB; DA018343 to PDC), F31 AG079606 to DMF and F31 DK136246 to JLK. This research was also funded in part through Aligning Science Across Parkinson’s (ASAP-025173 to TJM and PDC) through the Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Howard Hughes Medical Institute (HHMI; PDC). FS acknowledges support from the Human Frontier Science Program (LT000056/2020-C). JB acknowledges support by the Wellcome Leap Foundation. Imaging was supported by the Yale Center for Cellular and Molecular Imaging (both the fluorescence and electron microscopy facilities). We also thank the MS & Proteomics Resource at Yale University for providing the necessary mass spectrometers and the accompany biotechnology tools funded in part by the Yale School of Medicine and by the Office of The Director, National Institutes of Health (S10OD02365101A1, S10OD019967, and S10OD018034). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.