Western Blot for Cell Lysates: Complete Guide

Cell lysates are the most common sample type for western blotting, providing a comprehensive representation of cellular proteins. Proper preparation of cell lysates is crucial for obtaining reliable and reproducible results. This comprehensive guide provides optimized protocols for cell lysate preparation, including harvesting methods, lysis buffer selection, protein extraction, and optimization strategies for different cell types and experimental conditions.

Overview

Cell lysates contain the total protein complement of cells and are prepared by disrupting cells in appropriate lysis buffers. Cell lysates are ideal for western blotting because they:

Contain all cellular proteins in a single sample
Are relatively easy to prepare
Provide good representation of protein expression
Allow for normalization to total protein content
Can be prepared from various cell types
Are compatible with standard western blot protocols

Key considerations for cell lysate preparation:

Proper cell harvesting to maximize yield
Appropriate lysis buffer selection
Efficient protein extraction
Prevention of protein degradation
Accurate protein quantification
Proper storage conditions

Optimized cell lysate preparation is the foundation for successful western blot experiments.

Cell Harvesting Methods

Adherent Cells

Remove culture medium and wash cells with cold PBS
Add lysis buffer directly to cells on plate (recommended)
Or trypsinize cells and collect by centrifugation
Or scrape cells in cold PBS and collect
Keep all steps cold (4°C or on ice)
Minimize time between harvesting and lysis

Suspension Cells

Collect cell suspension from culture vessel
Centrifuge at 1,000-2,000 × g for 5 minutes at 4°C
Remove supernatant and wash pellet with cold PBS
Centrifuge again and remove PBS
Resuspend in appropriate volume of lysis buffer
Proceed with lysis protocol

Important Considerations

Harvest cells at appropriate confluency (70-90% for adherent cells)
Time treatments appropriately before harvesting
Keep cells cold throughout harvesting process
Count cells if normalization to cell number is needed
Process samples quickly to prevent degradation

Lysis Buffer Selection

RIPA Buffer (Most Common)

RIPA Buffer Composition:

50 mM Tris-HCl, pH 7.4
150 mM NaCl
1% NP-40 or Triton X-100
0.5% sodium deoxycholate
0.1% SDS
1 mM EDTA
Complete protease inhibitor cocktail

Advantages: Effective for most proteins, good extraction efficiency
Use for: General purpose, most cytosolic and membrane proteins
Considerations: May be too harsh for some sensitive proteins

NP-40 Lysis Buffer (Milder)

NP-40 Buffer Composition:

50 mM Tris-HCl, pH 7.4
150 mM NaCl
1% NP-40
1 mM EDTA
Complete protease inhibitor cocktail

Advantages: Milder, preserves some protein interactions
Use for: Sensitive proteins, protein complexes
Considerations: May not extract all proteins efficiently

SDS Lysis Buffer (Harsh)

SDS Buffer Composition:

50 mM Tris-HCl, pH 7.4
150 mM NaCl
1% SDS
1 mM EDTA
Complete protease inhibitor cocktail

Advantages: Strong extraction, good for difficult proteins
Use for: Membrane proteins, aggregated proteins
Considerations: Very harsh, may interfere with some applications

Protein Extraction Protocol

Standard Extraction Protocol

Harvest cells using appropriate method
Add lysis buffer with protease inhibitors (100-200 μL per 10⁶ cells)
Incubate on ice for 15-30 minutes
Vortex briefly every 5-10 minutes
Centrifuge at 10,000-15,000 × g for 10-15 minutes at 4°C
Collect supernatant (soluble proteins)
Determine protein concentration (BCA or Bradford assay)
Prepare samples for western blot

Optimization Tips

Use appropriate lysis buffer volume (not too much or too little)
Include complete protease inhibitor cocktail
Add phosphatase inhibitors if detecting phosphoproteins
Keep samples cold throughout process
Process samples quickly to prevent degradation
Vortex gently to avoid foaming

Optimization Strategies

Lysis Buffer Volume

Use 100-200 μL lysis buffer per 10⁶ cells
Adjust based on cell type and protein concentration needed
Too much buffer: dilute samples, may need concentration
Too little buffer: incomplete lysis, high viscosity
Test different volumes to find optimal

Protein Quantification

Use BCA or Bradford assay to determine protein concentration
Dilute samples if needed to fall within assay range
Prepare standard curve for accurate quantification
Normalize samples to equal protein content
Document protein concentrations for reproducibility

Sample Storage

Store lysates at -80°C for long-term storage
Aliquot samples to avoid repeated freeze-thaw cycles
Include protease inhibitors in storage buffer
Use samples as soon as possible for best results
Document storage conditions and duration

Troubleshooting

Low Protein Yield

Increase cell number or confluency
Optimize lysis buffer volume
Extend lysis time or use harsher lysis conditions
Check for incomplete cell harvesting
Verify lysis buffer composition

Protein Degradation

Include complete protease inhibitor cocktail
Keep samples cold throughout process
Process samples quickly
Check cell viability before harvesting
Use fresh inhibitors

High Viscosity

Dilute samples with lysis buffer
Centrifuge longer or at higher speed
Add DNAse if DNA is causing viscosity
Use less cells per volume of buffer