Cell Lysate Preparation for Western Blot

Preparing high-quality cell lysates is essential for successful western blotting. This detailed protocol provides step-by-step instructions for extracting proteins from cultured cells while maintaining protein integrity and preventing degradation.

Overview

Cell lysate preparation involves breaking open cells to release their protein content. The method must balance efficient protein extraction with preservation of protein structure and function. Key considerations include:

Maintaining protein integrity during lysis
Preventing protein degradation with protease inhibitors
Preserving post-translational modifications (e.g., phosphorylation)
Removing cellular debris and insoluble material
Accurately quantifying protein concentration

This protocol is optimized for both adherent cells (cells grown on culture dishes) andsuspension cells (cells grown in suspension). The basic principles are the same, but the handling methods differ slightly.

Materials Required

Reagents

• Ice-cold PBS (phosphate-buffered saline)
• Lysis buffer (RIPA, NP-40, or similar)
• Protease inhibitors (PMSF or commercial cocktail)
• Phosphatase inhibitors (NaF, Na3VO4)
• Protein quantification assay kit

Equipment

• Cell scraper (for adherent cells)
• Microcentrifuge tubes (pre-chilled)
• Refrigerated centrifuge
• Ice bucket
• Vortex mixer
• Sonicator (optional, for difficult cells)

Step-by-Step Protocol

Step 1: Wash Cells

Remove culture medium completely. Wash cells 2-3 times with ice-cold PBS to remove serum proteins and media components that could interfere with lysis or electrophoresis.

For adherent cells: Add PBS directly to the culture dish, swirl gently, and aspirate. Repeat 2-3 times. Ensure all PBS is removed completely to avoid dilution of lysis buffer.

For suspension cells: Pellet cells by centrifugation at 500-1000 × g for 5 minutes at 4°C. Remove supernatant and resuspend pellet in ice-cold PBS. Repeat washing 2-3 times.

Step 2: Prepare Lysis Buffer

Prepare lysis buffer fresh or use aliquoted buffer stored at -20°C. Common lysis buffers include:

RIPA buffer: Strong detergent, good for most proteins including membrane proteins
NP-40 buffer: Mild detergent, preserves protein-protein interactions
Triton X-100 buffer: Moderate strength, good for cytoplasmic proteins

Add inhibitors just before use:

Protease inhibitors: PMSF at 1 mM final concentration, or commercial protease inhibitor cocktail
Phosphatase inhibitors (for phosphorylated proteins): NaF at 10 mM, Na3VO4 at 1 mM

Keep buffer ice-cold throughout the process. Protease inhibitors degrade over time, so add them fresh.

Step 3: Add Lysis Buffer

Add appropriate volume of lysis buffer. Typical volumes:

100-200 μL per 10⁶ cells
100-150 μL per cm² of culture dish

For adherent cells: Add lysis buffer directly to the dish. Scrape cells using a cell scraper while the buffer is in the dish. Transfer the lysate to a pre-chilled microcentrifuge tube.

For suspension cells: Resuspend the cell pellet in lysis buffer. Pipette up and down several times to ensure complete resuspension. Transfer to a pre-chilled microcentrifuge tube.

Step 4: Incubate on Ice

Incubate on ice for 20-30 minutes with occasional gentle vortexing every 5-10 minutes. This allows time for the detergent to break open cell membranes and release proteins.

For difficult-to-lyse cells: You may need to extend incubation to 45 minutes or perform sonication (3-5 pulses of 10 seconds each on ice). Be careful not to over-sonicate as this can denature proteins.

Step 5: Centrifuge

Centrifuge at 12,000-14,000 × g for 15 minutes at 4°C. This removes:

Cell debris
Nuclei
Insoluble material
Unlysed cells

For some applications, you may need to centrifuge at higher speeds (20,000 × g) or perform sequential centrifugations to remove all insoluble material.

Step 6: Collect Supernatant

Carefully collect the supernatant without disturbing the pellet. Transfer to a new pre-chilled microcentrifuge tube.

Important: Avoid collecting any pellet material as it may contain insoluble aggregates, DNA, or other contaminants that can interfere with electrophoresis and cause smearing or background issues.

Step 7: Quantify Protein

Determine protein concentration using BCA, Bradford, or Lowry assay according to manufacturer's instructions. Always prepare a standard curve using BSA standards for accurate quantification.

Typical protein concentrations for cell lysates range from 1-10 mg/mL. If your sample is outside this range, you may need to:

Dilute concentrated samples before quantification
Concentrate dilute samples using protein concentrators
Adjust lysis buffer volume for future preparations

Lysis Buffer Selection

The choice of lysis buffer depends on your target protein and experimental requirements. Different buffers have different strengths and are suitable for different protein types:

RIPA Buffer

Best for: Most proteins, including membrane proteins and nuclear proteins

Composition: Contains strong detergents (SDS, NP-40, deoxycholate) that efficiently solubilize most proteins.

Note: May be too harsh for some protein-protein interactions. Check compatibility with your target protein.

NP-40 Buffer

Best for: Cytoplasmic proteins, preserving protein-protein interactions

Composition: Mild non-ionic detergent that preserves native protein structure.

Note: May not efficiently extract membrane proteins or nuclear proteins.

Triton X-100 Buffer

Best for: Moderate strength extraction, good balance

Composition: Non-ionic detergent with moderate strength.

Note: Good general-purpose buffer for most cytoplasmic proteins.

View Buffer Recipes →

Adherent vs Suspension Cells

While the basic principles of cell lysis are the same for both cell types, there are important differences in handling:

Adherent Cells

Grow attached to culture dish surface
Wash directly in the dish
Scrape cells in lysis buffer
Easier to control cell number
No centrifugation needed before lysis

Suspension Cells

Grow in suspension in culture medium
Require centrifugation to pellet
Wash by resuspending pellet
Resuspend in lysis buffer
May need to count cells for accurate normalization

Troubleshooting

Low Protein Yield

Possible causes:

Insufficient lysis buffer volume
Incomplete cell lysis (extend incubation or use sonication)
Protein degradation (use fresh inhibitors, work faster)
Proteins lost in pellet (check centrifugation conditions)

Protein Degradation

Solutions:

Use fresh protease inhibitors
Work quickly and keep everything on ice
Store lysates at -80°C immediately after preparation
Use appropriate lysis buffer for your cell type

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