Jun 04, 2025

COS-1 cell seeding, transfection, and lysis for protein expression assay V.1

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvokroxl4o/v1
  • 1NIMH
Christopher Bartley
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.5qpvokroxl4o/v1
Protocol Citation: Jailyn Izu 2025. COS-1 cell seeding, transfection, and lysis for protein expression assay. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvokroxl4o/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: May 15, 2024
Last Modified: March 26, 2026
Protocol  Integer ID: 99864
Keywords: lysis for protein expression, protein expression, general protocol for transfection, transfection, lysi
Funders Acknowledgements:
National Institute of Mental Health
Grant ID: ZIA MH002982
National Institute of Mental Health
Grant ID: R01MH122471
National Institute of Neurological Disorders and Stroke
Grant ID: U01NS120836
Foundation for the National Institutes of Health
Grant ID: Deeda Blair Research Initiative for Disorders of the Brain
Abstract
General protocol for transfection and lysate preparation
Materials
  • 6-well plate (Corning, #3506)
  • FuGene 4K Transfection Reagent (Promega, #E5911)
  • Opti-MEM Reduced Serum Medium (Thermo Fisher Scientific, #31985062)
  • Plasmid constructs
  • Dulbecco’s Modified Eagle Medium (DMEM) with GlutaMax (Thermo Fisher Scientific, #10569010)
  • FBS (Gibco, #26140-079)
  • Penicillin-streptomycin (Thermo Fisher Scientific, #15140122)
  • PBS, pH 7.4 (Thermo Fisher Scientific, #10010023)
  • M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, #78501)
  • Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, #78429)
Transfect cells and lyse
3d
Follow the general protocol:
Protocol
NAME

Transfecting COS-1 cells using FuGENE® 4K Transfection Reagent and cell lysis (M-PER) in a 6-well plate

CREATED BY
Jailyn Izu
Protocol-specific parameters:
  • For seeding cells in Step 1, seed 500,000 COS-1 cells per well (1 mL total volume per well) and incubate the plated cells in 37 °C with 5% CO2 for 24 hours 24:00:00 .
  • For Step 2, transfect cells at 80% confluence and use a 1:3 DNA-to-FuGENE (μg/μL) ratio, with 2 μg of total DNA for each sample.
  • For Step 3, transfect Flag wild-type, Flag-TBK1 N455S, or Flag-TBK1 M559R and co-transfect with EGFP (OmicsLink‱ Expression Clone EX-EGFP-M11; GeneCopoeia, Inc., Rockville, MD)
  • For Step 6, incubate transfected cells for 48 hours 48:00:00 , adding 1 mL of fresh media to each well after the first 24 hours.
  • For Step 9, add 500 µL / well M-PER reagent + 1X protease and phosphatase inhibitor to each well
3d