Feb 28, 2024
Immunoprecipitation for Flag-tagged proteins
- 1University of Ottawa
Protocol Citation: Jean-Louis Parmasad 2024. Immunoprecipitation for Flag-tagged proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7poxqgwz/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
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Created: February 28, 2024
Last Modified: February 28, 2024
Protocol Integer ID: 95879
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Abstract
Immunoprecipitation for Flag-tagged proteins for various biomolecular analyses.
Transfection and collection
Transfection and collection
Transfect HEK293T cells with 2µg of plasmid expressing Flag-tagged protein of interest using enhanced polyethyleneimine transfection reagent in Opti-MEM (Thermo Fisher Scientific, 31985070).
After 48h, collect cells and lyse with IP Lysis Buffer (25 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP4O, 5% Glycerol).
Prepare beads
Prepare beads
Defrost beads on ice. Wash 3x with IP Lysis Buffer.
IP
IP
Use 3% of the collected sample for the ‘Input’.
Incubate the remaining sample with Anti-FLAG M2 Magnetic beads (Millipore Sigma, M8823) for 1 h at 4 °C in a revolving rack.
Place tubes in magnetic rack and collect 3% the supernatant as the “Flow-through” sample. Aspirate the remaining flow-through.
Wash beads with IP lysis buffer.
Add 2x Laemmli buffer (Bio-Rad, 1610747, Hercules, CA, USA) with 20% 2-mercaptoethanol (Bio-Rad, 1610710) to the beads.
Vortex, then incubate for 15 min at 75 °C to elude proteins from the beads.
Place tubes in magnetic rack and collect elution.
Run all samples on SDS-PAGE gel and visualize with method of choice.