May 11, 2026
Purification of 3xFLAG-ATG2A from Expi293 Cells
- Devin Fuller1,2,
- Thomas Melia1,2
- 1Yale University;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, 20 MD
Protocol Citation: Devin Fuller, Thomas Melia 2026. Purification of 3xFLAG-ATG2A from Expi293 Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn5onqg5d/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: April 10, 2026
Last Modified: May 11, 2026
Protocol Integer ID: 314827
Keywords: protein purification, expi293f cells protocol, purification of the card8, expi293f cell, purification, protocol, atg2a from expi293 cells protocol, dpp9 complex from expi293f cell, expi293 cells protocol, purification of the nlrp1, expi293f cell, purification, dpp9 complex, nlrp1, atg2a
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
Protocol is largely the same as, "Purification of the NLRP1-DPP9 Complex from Expi293F Cells", with some small changes due to protocol differences in the Melia lab.
Materials
MATERIALS
Opti-MEM™ I Reduced Serum Medium, no phenol redThermo FisherCatalog #11058021
Expi293™ Expression MediumThermo FisherCatalog #A1435101
Expi293F™ CellsThermo FisherCatalog #A14527
vented PETG flasks (Thermo Scientific, Nalgene; 4115-0500)
Before start
Cells should be >95 % viable and doubling every 24 hr. Mantain cells with constant shaking at 100 RPM, 37°C, 5% CO2.
Need maxi-prepped DNA, ideally using an endotoxin-free kit.
Protein Expression
4d 19h 36m
Grow Expi293F cells (ThermoFisher, A14527) in Expi293™ Expression Medium (ThermoFisher, A1435101) to 2-3 million cells/mL at the desired culture volume, such as 100 mL. Choose the appropriate flask for the desired volume of cell culture to ensure appropriate gas exchange. If cells are grown slightly above 3 million cells/mL (up to 6 million cells/mL), dilute to 3 million cells/mL using fresh medium day-of. Otherwise, dilute below 1.5 million cells/mL and try again another day. Put cells back in the incubator once prepared, for now. Note that these cells require an orbital shaker to keep them in solution.
Note
Cells should be >95 % viable (e.g. with trypan blue, Thermofisher 15250061) and doubling every 24 h.
Warm Opti-MEM to RT. In the hood, transfer RT Opti-MEM 100 mL per 1L cells to a small sterile, autoclaved flask. For smaller culture volumes, falcon tubes can be used.
1m
Transfer endotoxin-free DNA encoding pCMV 3xFLAG-ATG2A into the Opti-MEM flask. Swirl to mix.
5m
Add polyethylenimine (PEI, 300 µL per 100 mL cells at 1 mg/mL or 1.5 mL per 1L cells at 2 mg/mL ). Swirl to mix. Incubate in the hood 30-45 min 00:30:00 . See the following resource for preparing PEI: https://www.addgene.org/protocols/transfection/
30m
In the cell culture hood, slowly pour Opti-MEM-DNA-PEI mixture into the flask containing cells (from step 1) while gently shaking. Return flask to the incubator, and incubate 18:00:00 .
18h
Add sterile-filtered valproic acid (VPA, 700 uL per 100 mL.of cells, 500 mM, prepared from Sigma P4543) into the flask with the transfected cells. This step boosts protein production. Return to the shaker for 48-72 hrs.
4d
Harvest cells by centrifugation 2000 rpm, 4°C, 00:20:00 . Discard the supernatant. The resulting pellet can be stored at -80C until protein purification. Sterile technique is no longer required. Note that a ring of dead cells will be present in a rim of the upper flask.
25m
Protein Purification
5d 1h 26m
Resuspend the cell pellets in 10 mL of pre-chilled purification buffer A (500 mM NaCl, 20 mM HEPES, pH: 8.0, 10% glycerol, 1 mM TCEP), supplemented with 1x EDTA-free protease inhibitor cocktail (Roche; 11873580001) per 100 mL of cells.
Lyse the cells by subjecting the pellet resuspensions to 6 freeze/thaw cycles in liquid nitrogen.
Clarify the lysates by centrifugation at 18,500 x G for 30 min.
Prepare affinity columns by washing 150 μL of anti-Flag M2 agarose resin per 100 mL of cells with buffer A.
Transfer the supernatants to the columns and incubate under mild agitation for 2 hours at 4 °C
Pass the lysate through the gravity filtration column and then use four column volumes of buffer A to wash the beads.
Incubate the beads with 2.5 mM ATP and 5 mM Mg2+ in buffer A overnight at 4 °C to remove chaperones.
Wash the columns two more times with buffer A.
Elute the protein using buffer A supplemented with 50 μL of 1 mg/mL 3x-Flag (DYKDDDDK) peptide (ApexBio Technology; A6001) per 100 mL of initial culture volume.
Determine protein concentration against a BSA standard curve by Coomassie staining prior to subsequent assays.
Flash freeze the protein samples in liquid nitrogen for storage at -80 °C.
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.