Apr 29, 2026
Protein extraction and quantification from mouse tissues or cells
- Sriparna Mukherjee1,2,3,4,
- Amandine EVEN1,2,3,5,4,
- Louis-Éric Trudeau1,6,2,3,5,4
- 1Department of pharmacology and physiology, Faculty of Medicine, Université de Montréal;
- 2Neural Signaling and Circuitry research group (SNC);
- 3Center for Interdisciplinary Research on the Brain and Learning (CIRCA);
- 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
- 5Institut Courtois d’innovation biomédicale;
- 6Department of neuroscience and physiology, Faculty of Medicine, Université de Montréal
Protocol Citation: Sriparna Mukherjee, Amandine EVEN, Louis-Éric Trudeau 2026. Protein extraction and quantification from mouse tissues or cells. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjkwqxgk5/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: April 20, 2026
Last Modified: April 29, 2026
Protocol Integer ID: 315387
Keywords: protein extraction, quantification from mouse tissue, cells this protocol, quantification from tissue, ripa buffer, mouse tissue, extraction, cell, ezq kit, laemmli buffer
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000525
Abstract
This protocol describes protein extraction and quantification from tissue (with RIPA buffer and BCA assay) or cells (with Laemmli buffer and EZQ kit) .
Guidelines
Avoid unnecessary freeze-thaw cycles for tissue and cells lysate samples.
Materials
From tissue
**Tissue lysis**
1. RIPA buffer (Fisher Scientific, Cat# PI89900)
2. Protease inhibitor cocktail (Sigma-Aldrich, Cat# P840)
3. Phosphatase inhibitor cocktail (Sigma-Aldrich, Cat#P0044)
4. Homogenizer - Ultra-Turrax T8 (Ika-Werke, Germany)
5. Centrifuge capable of 12000 X g
**Protein quantification**
1. BCA Protein Assay Kit (Thermo Scientific Pierce, Cat# PI23227)
2. Bovine Serum Albumin (BSA) Quick Start Kit (Biorad)
3. Plate reader 562nm
**Denaturation**
1. B-Me
2. DTT
3. Dry bath
From cells
**Tissue lysis and denaturation**
1. 4x Laemmli Sample Buffer (Biorad, Cat#1610747)
2. Protease inhibitor cocktail (Sigma-Aldrich, Cat# P840)
3. Phosphatase inhibitor cocktail (Sigma-Aldrich, Cat#P0044)
4. Benzonase Nuclease (Sigma, Cat#E1014-25KU)
5. B-Me
6. Dry bath
**Protein quantification**
1. EZQ Protein Quantitation Kit (Invitrogen, Cat#R33200)
2. Ovalbumin
3. Acetic acid
4. Methanol
5. Plate reader 618nm
6. Hair dryer with cold function
Safety warnings
IMPORTANT: All the experiment have to be done on ice to avoid protein degradation.
Before start
- Pre-label tubes and prepare gels tissue thawing.
- Keep tissue, lysates, and centrifuge steps in cold temperature.
- Prepare fresh RIPA buffer supplemented with protease inhibitor cocktail immediately before use.
From tissue
1h 25m
Protein extraction:
Prepare the lysis buffer mixing RIPA buffer with protease inhibitor cocktail (1/2000).
If necessary, add phosphatase inhibitor cocktail (1/2000).
Dissect the tissue and transfer immediately to cold lysis buffer to homogenize.
If homogenization cannot be carried out directly, the tissue can be frozen in a dry tube with dry ice and stored at -80 °C for later homogenization.
For brain samples, mice were perfused with 1X PBS before dissection.
Homogenize each piece of tissue in the lysis buffer using an Ultra-Turrax T8 homogenizer.
Use a consistent lysis volume across samples within the experiment.
For example, 700 µL of lysis buffer is used per striatum and 300 µL per mesencephalon
Clarify lysate:
Centrifuge homogenates at 12000 rpm, 4°C, 00:30:00 .
Carefully collect the supernatant without disturbing insoluble material.
30m
Protein quantification:
Measure protein concentration using the BCA assay:
Add 20 µL of autoclaved MilliQ Water to a 96-well flat-bottom plate.
Add 5 µL of sample.
For the calibration curve, use the Bovine Serum Albumin (BSA) Quick Start Kit.
Prepare a 1/50 CuSO4 solution in BCA buffer.
Add 200 µL of this BCA/CuSO4 solution to each well.
Place the plate in aluminum foil and incubate for 00:25:00 at 37 °C .
25m
Read with a plate reader at 562nm.
Protein can be stored at -20 °C at this step or at -80 °C for long-term storage.
If samples will be used to measure secreted proteins such as cytokines, each lysate concentration is adjusted in order to have 30 μg total protein per tube. Then samples can be frozen at -80 °C and used latter for the cytokine assay.
If samples will be used in Western Blot, each lysate will be denatured with DTT or β-mercaptoethanol (B-Me):
Prepare denaturing solution
- DTT: 500 µL loader + 7 mg DTT
- B-Me: 500 µL loader + 10 µL B-Me
Add ¼ of the sample in DTT or B-Me
Heat for
- 00:30:00 at 37 °C for DTT
- 00:05:00 at 95 °C for B-Me
And chill on ice
Protein extracts can be stored at -20 °C at this step, at -80 for long-term storage
30m
Normalize all samples to a common concentration suitable for gel loading.
From cells
1h 11m
Protein extraction and denaturation:
Prepare the lysis solution mixing 1 mL 4x Laemmli Sample Buffer in 3 mL MilliQ water and adding β-mercaptoethanol and protease inhibitor cocktail (1/2000).
If necessary, add phosphatase inhibitor cocktail (1/2000).
Rinse cells with 1X PBS.
Cells can be stored without media at -80 °C at this step.
Add 100 µL of lysis solution per million of cells per well.
Add 1 µL of benzonaze per 100 µL sample to reduce the viscosity of the solution.
Microglia and astrocytes give viscous solutions, so, more benzonaze is needed (1 µL per 50 µL of sample).
Cells can be stored at -80 °C at this step.
Scrape the bottom of the well with a P10 tip until all the material is collected and a liquid solution is obtained.
Transfer the sample to an eppendorf tube.
Heat at95 °C during 00:10:00 and put in ice to chill.
Proteins can be stored at -20 °C at this step.
10m
Protein quantification with a EZQ Kit:
Prepare dilutions of the ovalbumin stock solution:
Prepare standards by making serial dilutions of the 10 mg/mL ovalbumin stock solution. The dilution buffer should be the same as that used for the experimental samples (Laemmli lysis solution). At least five concentrations should be used to cover the range expected for the experimental samples. The full effective protein concentration range for this assay is ~0.02 mg/mL –5 mg/mL .
DONT FORGET THE BLANK (Laemmli lysis solution only)
Load 1 µL of ovalbumin standard solutions and samples on the membrane from the EZQ kit.
Perform triplicates for each.
Be careful to not damage the membrane - avoid to touch it when loading the samples.
Dry with a hair dryer without heat 00:01:00
00:10:00 if done without dryer
11m
Incubate the membrane in a methanol bath (30 mL -40 mL ) during 00:05:00 with agitation (under a fume hood).
5m
Dry with a hair dryer without heat 00:01:00
00:10:00 is needed for drying if not using a dryer
Place the membrane in 30 mL EZQ solution, protect from light, and incubate 00:30:00 under agitation (under a fume hood).
30m
Wash 3 times 00:02:00 with washing buffer (10% methanol, 7% acetic acid)
15m
Dry with a hair dryer without heat 00:01:00
00:10:00 is needed for drting if not using a dryer
Read with a plate reader at 618nm.
Proteins can be stored at -20 °C at this step.