Mar 29, 2025
Protein Immunoprecipitation (IP) from HEK293 Cells or iNeurons
- Miguel A. Gonzalez-Lozano1,
- Harper JW2
- 1Harvard Medical School;
- 2harvard university
External link: https://doi.org/10.1038/s41586-025-09059-y
Protocol Citation: Miguel A. Gonzalez-Lozano, Harper JW 2025. Protein Immunoprecipitation (IP) from HEK293 Cells or iNeurons. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjn5zpgk5/v1
Manuscript citation:
Gonzalez-Lozano, M.A., Schmid, E.W., Miguel Whelan, E. et al. EndoMAP.v1 charts the structural landscape of human early endosome complexes. Nature 643, 252–261 (2025). https://doi.org/10.1038/s41586-025-09059-y
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: October 18, 2024
Last Modified: March 29, 2025
Protocol Integer ID: 110315
Keywords: ASAPCRN, endogenous immunoprecipitation, protein immunoprecipitation, extracts from either hek293 cell, hek293 cell, protein, endogenous protein, ineurons this protocol, downstream analysis by mass spectrometry, mass spectrometry, es cell, cell
Funders Acknowledgements:
aligning science across parkinson's
Grant ID: 000282
aligning science across parkinson's
Grant ID: 024268
Abstract
This protocol described the methods used to isolate HA-tagged or endogenous proteins by co-IP followed by downstream analysis by mass spectrometry. The protocol uses extracts from either HEK293 cells or induced neurons derived from ES cells.
Materials
Pierce Anti-HA Magnetic Beads (Thermo Scientific, 88837)
n-Dodecyl β-D-maltoside (DDM, Gold Biotechnologies, DDM5)
protease inhibitor cocktail (Roche, 4906845001)
Anti-FLAG M2 Magnetic Beads (Sigma Millipore, M8823)
anti-TMEM230 (Origene, TA504888)
Pierce Protein A/G magnetic beads (Thermo Scientific, 88802)
Laemmli buffer [4% SDS, 20% glycerol, 0.004% bromphenol blue, 0.125M Tris-Cl, pH 6.8, 10% 2-mercaptoethanol (or DTT) (add immediately before use)]
Triethylammonium bicarbonate buffer (TEAB, Sigma-Aldrich, T7408)
Troubleshooting
Protein Extraction from HEK293 Cells or iNeurons
Prepare protein extracts from a 10 cm culture dish of HEK293 cells or a 15 cm dish of iNeurons for each replicate.
Collect cells directly in lysis buffer containing 0.5% n-Dodecyl β-D-maltoside (DDM), 25 mM HEPES (pH 7.4), 150 mM NaCl, and a protease inhibitor cocktail.
Extract proteins by incubating the cell lysate for 1 hour at 4°C in rotation.
Centrifuge the lysate at 20,000 x g for 20 minutes at 4°C. Repeat this centrifugation step to ensure complete clarification of the supernatant.
Immunoprecipitation
Collect the supernatant and incubate with magnetic beads depending on the protein tag:
- For HA-tag, 15 μL α-HA magnetic beads (Pierce) for 2 hours at 4°C.
- For Flag-tag, 25 μL α-Flag magnetic beads (Sigma) for 2 hours at 4°C.
- For endogenous antibodies, add 5 μg of the antibody to the supernatant and incubate overnight at 4°C before proceeding to bead incubation. Incubate with 15μL magnetic A/G for 2 hours at 4°C. In this example, antibodies against TMEM230 are used.
Separate the beads from the solution using a magnetic stand.
Wash the beads four times with 1 mL washing buffer containing 0.1% DDM, 25 mM HEPES (pH 7.4), and 150 mM NaCl.
Elute the bound proteins from the beads by adding 30 μL of either:
- 1.5X Laemmli buffer for immunoblotting
- 1.5X S-Trap lysis buffer (7.5% SDS, 150 mM Triethylammonium bicarbonate or TEAB, pH 8.5) for mass spectrometry (MS) analysis.