Apr 15, 2026

Preparation of total cell lysates

DOI
https://dx.doi.org/10.17504/protocols.io.eq2ly66wpgx9/v1
  • 1KU Leuven;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Sarah van Veen
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2ly66wpgx9/v1
Protocol Citation: Sarah van Veen, Peter Vangheluwe 2026. Preparation of total cell lysates. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly66wpgx9/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: December 13, 2024
Last Modified: April 15, 2026
Protocol  Integer ID: 115371
Keywords: ASAPCRN, ATP13A4, Cell lysate, preparation of total cell lysate, preparing cell lysate, total cell lysate, cell lysate, preparation, cell, step method
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Abstract
This protocol outlines a step-by-step method for preparing cell lysates.
Materials
Materials
  • Dulbecco's Phosphate Buffered Saline without calcium chloride and magnesium chloride (DPBS) (Cat# 14190144, GIBCO)
  • TrypLE (Cat# 12604021, GIBCO)
  • RIPA lysis and extraction buffer (Cat# 89901, Thermo Fisher)
  • SigmaFastTM Protease Inhibitor Cocktail (Cat# S8830, Sigma)
  • Pierce BCA protein assay kit (Cat# 23225, Thermo Fisher)

Equipment
  • Centrifuge
Safety warnings
Follow institutional guidelines for the disposal of biological and chemical waste.
Seed cells into a 10 cm dish at a density appropriate to achieve 70–80% confluence by the following day. Allow the cells to attach and grow for approximately 24:00:00 .

1d
Collect medium.
Wash cells with DPBS.
Incubate cells with TrypLE, 37 °C .

Collect cells.
Centrifuge for 00:05:00 at 400 x g, 4°C .

5m
Decant supernatant.
Wash pellet with DPBS.
Centrifuge for 00:05:00 at 400 x g, 4°C .
5m
Decant supernatant.
Add RIPA lysis buffer (supplemented with protease inhibitors).
Incubate for at least 00:30:00 On ice , or alternatively store at -20°C until further processing.

30m
Centrifuge 15000 x g, 4°C, 00:30:00 to pellet DNA and non-solubilized material.

30m
Keep supernatant.
Determine protein concentration via BCA assay.