JNAR Protocol

RIPA-Based Total Protein Extraction from Mammalian Cells

Núria Crua· Postdoc· University of Lausanne (UNIL)

Published Jul 27, 2026

~40m total· 0 contributions

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JNAR-ID: JNAR-40363742

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PublishedLast updated Jul 27, 2026

Objective

This protocol describes the extraction of total proteins from mammalian cells using RIPA buffer. The resulting protein lysates are suitable for downstream applications such as Western blotting and total protein analysis.

Classification

Sample typeProteinCells

TechniqueProtein Purification

Experimental contextIn vitro

Biological modelPrimary CellsCell Lines

Reproducibility status

Reproducible

No reproducibility blockers or risks reported. The protocol runs end-to-end.

11 healthy

⏱ Total 40 min2 sections11 steps

Buffer Preparation

Prepare the lysis buffer immediately before use.

Critical stepProtease and phosphatase inhibitors must be added immediately before use to preserve protein integrity.

Supplemented RIPA Buffer (1 mL)

Reagent	Volume
RIPA Buffer	860 µL
25× Protease Inhibitor Cocktail	40 µL
10× Phosphatase Inhibitor	100 µL
Final Volume	1000 µL

Prepare enough supplemented RIPA buffer for all samples plus one additional sample to compensate for pipetting losses.

Procedure

40 min

Collect approximately 1 × 10⁶ mammalian cells per sample.

3.1

Suspension cells: Pellet the cells by centrifugation.

3.2

Adherent cells: Remove the culture medium and wash the cell monolayer directly in the culture vessel.

Wash the cells with 500 µL of ice-cold PBS.

4.1

Centrifuge (if applicable): 500 × g, 5 minutes, 4°C.

4.2

Carefully remove all remaining PBS.

Add approximately 100–200 µL of supplemented ice-cold RIPA buffer per 1 × 10⁶ cells. Adjust the volume according to pellet size and desired protein concentration.

5.1

Resuspend the pellet completely by gentle pipetting.

5.2

For adherent cultures, distribute the RIPA buffer evenly over the culture surface and detach the cells using a cold cell scraper or repeated pipetting.

Incubate the lysates for 20 minutes on ice. Mix gently every few minutes by pipetting or gentle agitation.20 min

Critical stepMaintaining low temperature throughout the procedure is essential for preserving protein integrity.

Transfer the complete lysate into pre-cooled microcentrifuge tubes.

TipIf the lysate appears highly viscous, gently homogenize by repeated pipetting. Avoid vigorous vortexing, which may promote protein denaturation or foaming.

Centrifuge the lysates at 14,000 × g for 20 minutes at 4°C to remove insoluble cellular debris.20 min

Carefully transfer the clear supernatant into a new pre-cooled tube. Avoid disturbing the pellet.

Critical stepThe supernatant contains the soluble protein fraction; disturbing the pellet may contaminate the lysate with debris.

Remove an aliquot (typically 10–20 µL) for protein quantification using a BCA assay. Use a buffer blank prepared with the same supplemented RIPA buffer.

Aliquot the remaining protein lysate for storage.

Critical stepAvoid repeated freeze–thaw cycles to preserve protein integrity.

11.1

Store at −80°C for long-term storage.

11.2

Store at −20°C for short-term use.