Dec 01, 2025
Immunoprecipitation with GFP-Trap Beads
- 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 N
Protocol Citation: Devin M. Fuller, Thomas Melia 2025. Immunoprecipitation with GFP-Trap Beads. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3k6b7v25/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: April 09, 2025
Last Modified: December 01, 2025
Protocol Integer ID: 126468
Keywords: tagged protein, trap bead, protein, experiments with gfp, gfp
Funders Acknowledgements:
National Institutes of Health
Grant ID: R01 GM100930
National Institutes of Health
Grant ID: R35 GM153482
National Institutes of Health
Grant ID: DA018343
National Institutes of Health
Grant ID: F31 AG079606
National Institutes of Health
Grant ID: F31 DK136246
Aligning Science Across Parkinson’s
Grant ID: ASAP-025173
Human Frontier Science Program
Grant ID: LT000056/2020-C
Abstract
A protocol to perform co-immunoprecipitation (CoIP) experiments with GFP tagged proteins.
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)
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)
Other reagents:
GFP-Trap beads (Proteintech, gtma-20)
Troubleshooting
Cell culture and treatments
10m
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 on 15 cm plates at such density so as to be approximately 95% confluent at the time of lysis.
If transfecting the sample, do so when the cells are approximately 70-80% confluent. Prepare the following reagents for transfection with lipofectamine 3000:
For transfected plasmids, each 15 cm plate requires 10 µg of each target DNA construct. Prepare a master mix consisting of 10 µg of DNA, 700 µL of opti-mem low serum medium, and 20 µL of P3000 reagent per well. Prepare an additional mix consisting of 700 µL of opti-mem and 20 µL of lipofectamine 3000 per well. Combine the DNA + P3000 and lipofectamine solutions together, and allow to incubate for 00:10:00 at Room temperature .
10m
Cell lysis and sample preparation
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.
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 .
Determine protein concentration in sample using Bradford Assay.
Immunoprecipitation
2h 10m 5s
Prepare GFP-Trap beads for the IP reaction:
Pipette 25 µL of the GFP-Trap bead slurry into eppendorf tubes for each reaction.
Pipette 500 µL of lysis buffer onto the bead slurry. Mix the tube by inverting five times.
Precipitate the beads using a magnetic rack. Aspirate the lysis buffer, including the buffer trapped at the top of the tube.
Pipette 500 µL of lysis buffer onto the bead slurry. Mix the tube by inverting five times.
Precipitate the beads using a magnetic rack. Aspirate the lysis buffer, including the buffer trapped at the top of the tube.
Pipette equal amounts of lysate (by protein level) onto the washed GFP-Trap beads. Adjust the volume up to 500 µL .
Incubate the samples at 4 °C for 02:00:00 , rotating.
2h
Perform the following washes at Room temperature :
Aspirate the flow through. Pipette 500 µL of lysis buffer onto the bead slurry. Mix the tube by inverting five times. Precipitate the beads using a magnetic rack. Aspirate the lysis buffer, including the buffer trapped at the top of the tube.
Pipette 500 µL of lysis buffer onto the bead slurry. Mix the tube by inverting five times. Precipitate the beads using a magnetic rack. Aspirate the lysis buffer, including the buffer trapped at the top of the tube.
Pipette 500 µL of lysis buffer onto the bead slurry. Mix the tube by inverting five times. Precipitate the beads using a magnetic rack. Aspirate the lysis buffer, including the buffer trapped at the top of the tube.
Pipette 500 µL of lysis buffer onto the bead slurry. Mix the tube by inverting five times. Precipitate the beads using a magnetic rack. Aspirate the lysis buffer, including the buffer trapped at the top of the tube.
Pipette 500 µL of lysis buffer onto the bead slurry. Mix the tube by inverting five times. Precipitate the beads using a magnetic rack.
Briefly centrifuge the samples for 00:00:05 . Aspirate the buffer.
5s
Elute the protein by heating the samples at 95 °C in 2x LDS loading buffer for 00:10:00 . Pipette the eluted sample into a new eppendorf tube or directly in to the gel.
10m
Gel electrophoresis and immunoblotting
2h 10m 5s
Prepare samples at desired concentration, add 1 mM DTT and 1x LDS loading buffer.
Incubate samples at 95 °C for 00:05:00 .
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.
Block membrane with 5% BSA in PBST for 01:00:00 at 22 °C .
Incubate membrane with primary antibodies in 5% BSA and 0.03% sodium
azid in PBST Overnight at 4 °C .
Note
NOTE: Optimal primary antibody incubation time and temperature can be determined empirically for a given primary antibody
Wash membrane with PBST for five minutes. Repeat a total of 3 times.
Wash membrane for 00:05:00 with PBST (1/3).
Wash membrane for 00:05:00 with PBST (2/3).
Wash membrane for 00:05:00 with PBST (3/3).
Incubate membrane with secondary antibodies (1:5,000) in 5% milk in PBST for 01:00:00 at 22 °C .
Wash membrane with PBST. Repeat a total of 3 times.
Wash membrane for 00:05:00 with PBST (1/3).
Wash membrane for 00:05:00 with PBST (2/3).
Wash membrane for 00:05:00 with PBST (3/3).
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.