Dec 03, 2024

Public workspaceHA Immunoprecipitation

The Journal of Cell Biology
DOI
dx.doi.org/10.17504/protocols.io.3byl4wbo8vo5/v1
  • 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
Amanda Bentley-DeSousa
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DOI: dx.doi.org/10.17504/protocols.io.3byl4wbo8vo5/v1
Protocol Citation: Amanda Bentley-DeSousa, Shawn Ferguson 2024. HA Immunoprecipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wbo8vo5/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: 113417
Keywords: Immunoprecipitation, HA
Funders Acknowledgements:
Aligning Science Across Parkinson’s Disease
Grant ID: ASAP-000580
Abstract
This protocol details steps taken to perform HA tag immunoprecipitations.
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)]
anti-HA beads (Thermo Fisher Scientific, 88837)
TBST [10 mM Tris Base (American Bio AB02000-05000), 150 mM NaCl (Sigma-Aldrich, 3624-05), 0.1% Tween 20 (Sigma-Aldrich, P7949)]
Day 0
Day 0
Split a ~90% confluent 15-cm dish of RAW 264.7 cells into 2 15-cm dishes.
Day 1
Day 1
1h 19m
1h 19m
Add the indicated compound at the provided concentration and time point in fresh DMEM media containing FBS and P/S.
Proceed to next step if not treating cells.
Wash each dish 2X with cold PBS on ice.
Remove the PBS, scrape each well with Amount700 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 Duration00:05:00 .

5m
Centrifuge Centrifigation14000 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.
To generate the Whole Cell Lysate: mix an aliquot of the supernatant/lysate 1:1 with Laemmli Buffer and heat at Temperature95 °C for Duration00:03:00 .

3m
Laemmli mBuffer: 80 mM Tris-HCl pH 6.8, 25.4% Glycerol, 2.67% SDS, and Bromophenol Blue supplemented with 6.187% fresh B-mercaptoethanol.
Create a mastermix of anti-HA beads (Amount10 µL of beads per sample) and wash 3X with ice-cold lysis buffer.

Immunoprecipitate lysates using Amount10 µL anti-HA beads by incubating rotating end-over-end for Duration01:00:00 at Temperature4 °C .

1h
Ensure the same amount of protein was used for each sample and where needed, supplement samples with lysis buffer to ensure the same volume is used.
Wash beads 3X with 0.1% TBST.
TBST: 10 mM Tris Base, 150 mM NaCl, 0.1% Tween 20.
Wash beads 1X with mqH2O and transfer to new Eppendorf tubes.
Elute samples by incubating beads with Amount40 µL Laemmli buffer and warming at Temperature95 °C for Duration00:03:00 .

3m
Transfer the eluant to a new Eppendorf tube and supplement with 6.187% B-mercaptoethanol.
Subject samples to electrophoresis and immunoblotting as in Protein Lysate and Immunoblotting.