May 29, 2024
Quick protocol for protein extraction from adherent fish-derived fibroblasts
- Joao M Moreno1,2,
- Vitor C Sousa1,
- Romana Santos2
- 1cE3c – Centre for Ecology, Evolution and Environmental Changes & CHANGE – Global Change and Sustainability Institute, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal;
- 2MARE – Centro de Ciências do Mar e do Ambiente (MARE) & ARNET—Aquatic Research Network, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal
Protocol Citation: Joao M Moreno, Vitor C Sousa, Romana Santos 2024. Quick protocol for protein extraction from adherent fish-derived fibroblasts. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71ydkgwz/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 16, 2024
Last Modified: May 29, 2024
Protocol Integer ID: 99955
Keywords: Protein extraction, Protein purification, Cell culture
Funders Acknowledgement:
Fundação para a Ciência e a Tecnologia
Grant ID: SFRH/BD/143199/2019
Fundação para a Ciência e a Tecnologia
Grant ID: PTDC/BIA-EVL/4345/2021
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Abstract
We present a rapid and efficient protocol for extracting total proteins from adherent fish-derived fibroblasts, specifically optimized for applications in Western blotting and mass spectrometry. The method involves directly dissociating cells from the culture flask into a lysis buffer (e.g. RIPA buffer) followed by centrifugation to collect the total soluble proteins. The use of buffers like RIPA ensures comprehensive solubilization of cellular proteins while preserving their integrity and functionality. This straightforward and reproducible protocol yields high-quality protein extracts suitable for various downstream analytical techniques. Its simplicity and reliability make it an invaluable tool for researchers studying proteomics using fish cell culture.
Materials
Solutions
- 1X Phosphate Buffered Saline (PBS)
- RIPA lysis buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, sterile-filtered.
Materials
- 2 mL or 1.5 mL microcentrifuge tubes
- Cell scrappers
- Pipettes and pipette tips
- Serological pipettes (variable volume)
- Pasteur pipettes
Equipments
- Refrigerated microcentrifuge
- Flow hood chamber
Carefully remove all culture media from the flask and add enough ice-cold 1X PBS to wash the cells
Carefully remove the ice-cold 1x PBS and add ice-cold lysis buffer (RIPA buffer) according to the estimated number of cells:
1 mL for cells (roughly a T75 flask).
Use a cell scraper to dissociate the cells from the bottom of the flask.
Resuspend the cells in the lysis buffer and transfer the suspension to a microcentrifuge tube.
Agitate for 20 minutes at 4ºC.
Centrifuge at 13,000 x g for 20 min at 4°C.
Carefully transfer the supernatant containing the soluble protein to a new tube and keep on the ice. Discard the pellet.
Note: The protein solution can be kept in the freezer for longer storage periods until further use.