Tissue Sample Preparation for Western Blot

Preparing protein extracts from tissue samples requires more vigorous methods than cell lysis. This comprehensive protocol provides detailed instructions for homogenizing tissues and extracting proteins while maintaining protein integrity and preventing degradation.

Overview

Tissue homogenization is more challenging than cell lysis because tissues contain extracellular matrix, connective tissue, and other structural components that require mechanical disruption. The key differences from cell lysis include:

Requires mechanical homogenization (not just detergent)
May need additional steps to remove lipids or connective tissue
Typically yields higher protein concentrations (5-20 mg/mL)
May require filtering to remove debris
Some tissues need special handling (e.g., brain, muscle, liver)

This protocol covers general tissue homogenization methods that can be adapted for most tissue types. Specific tissues may require modifications, which are discussed in the Different Tissue Types section.

Materials Required

Reagents

• Fresh or frozen tissue samples
• Ice-cold lysis buffer (RIPA or similar)
• Protease inhibitors
• Phosphatase inhibitors
• Liquid nitrogen (for flash-freezing)
• Protein quantification assay kit

Equipment

• Mechanical homogenizer
• Mortar and pestle (alternative)
• Bead-beating system (alternative)
• Scalpel or razor blade
• 0.45 μm syringe filter
• Refrigerated centrifuge
• Pre-chilled microcentrifuge tubes

Step-by-Step Protocol

Step 1: Collect and Prepare Tissue

Collect fresh tissue and immediately place on ice or flash-freeze in liquid nitrogen. For frozen tissues, keep frozen until ready to process to prevent protein degradation.

Flash-freezing: For optimal protein preservation, flash-freeze tissue in liquid nitrogen immediately after collection. Store at -80°C until ready to process.

Cut tissue into small pieces (2-3 mm³) using a scalpel or razor blade on a chilled surface. Smaller pieces homogenize more efficiently and yield better protein extraction.

Important: Work quickly and keep tissue cold throughout preparation to prevent protein degradation.

Step 2: Homogenize Tissue

Homogenize tissue in ice-cold lysis buffer. Typical buffer volume is 10-20 volumes per weight of tissue (e.g., 100 mg tissue in 1-2 mL buffer). The exact ratio depends on tissue type and desired protein concentration.

Homogenization methods:

Mechanical homogenizer: Most efficient, use multiple short bursts (10-15 seconds each) with cooling intervals
Mortar and pestle: Good for small samples, requires liquid nitrogen for frozen tissues
Bead-beating system: Effective for tough tissues, use appropriate bead size

For tough or fibrous tissues, perform homogenization in multiple short bursts with cooling intervals to prevent overheating and protein denaturation.

Step 3: Incubate Homogenate

Incubate homogenate on ice for 30 minutes with occasional vortexing. This allows time for:

Detergent to solubilize membrane proteins
Protease inhibitors to prevent degradation
Complete protein extraction from tissue fragments

For fibrous tissues (e.g., muscle, tendon), extend incubation to 45-60 minutes. Some tissues may benefit from brief sonication (3-5 pulses of 5 seconds each on ice) to improve extraction.

Note: Avoid excessive sonication as it can denature proteins and cause foaming.

Step 4: Centrifuge

Centrifuge at 12,000-14,000 × g for 20 minutes at 4°C. This removes:

Tissue debris
Connective tissue fragments
Insoluble material
Lipid droplets (partially)

For tissues with high lipid content (e.g., adipose tissue, brain), you may need to centrifuge at higher speeds (20,000 × g) or perform an additional centrifugation step to remove lipids.

Step 5: Filter (If Needed)

If the supernatant contains visible debris or is cloudy, filter through a 0.45 μm syringe filter or centrifuge again. Some protocols recommend filtering through cheesecloth or fine mesh for initial debris removal.

Filtering tips: Pre-wet the filter with lysis buffer to prevent protein loss. For very viscous samples, you may need to dilute before filtering.

Step 6: Quantify Protein

Determine protein concentration using BCA, Bradford, or Lowry assay. Tissue extracts typically have higher protein concentrations (5-20 mg/mL) than cell lysates.

Dilute samples as needed to fall within the linear range of your quantification assay. Always prepare a standard curve using BSA standards for accurate quantification.

Important: Some tissue extracts may contain components that interfere with protein assays. If you get unexpected results, try a different assay method or dilute the sample further.

Homogenization Methods

Mechanical Homogenizer

Best for: Most tissue types, especially soft tissues

Advantages: Fast, efficient, reproducible

Protocol: Use multiple short bursts (10-15 seconds each) with cooling intervals. Keep samples on ice between bursts.

Tips: Use appropriate probe size for your sample volume. Clean probe thoroughly between samples.

Mortar and Pestle

Best for: Small samples, frozen tissues

Advantages: Gentle, good for preserving protein structure

Protocol: Pre-chill mortar and pestle. For frozen tissues, grind in liquid nitrogen, then add lysis buffer.

Tips: Keep everything cold. Work quickly to prevent thawing.

Bead-Beating System

Best for: Tough tissues, high-throughput applications

Advantages: Efficient, can process multiple samples simultaneously

Protocol: Use appropriate bead size and material. Follow manufacturer's recommendations for speed and time.

Tips: Use appropriate buffer volume to ensure proper bead movement. Cool samples between runs.

Different Tissue Types

Different tissue types may require specific modifications to the standard protocol:

Brain Tissue

High lipid content - may need additional centrifugation or lipid removal steps
Very soft - gentle homogenization sufficient
High protein content - may need dilution before quantification
Consider using specialized buffers for membrane proteins

Muscle Tissue

Fibrous and tough - requires vigorous homogenization
May need extended incubation time (45-60 minutes)
Consider using stronger lysis buffers (RIPA)
May benefit from sonication

Liver Tissue

High protein content - very efficient extraction
May contain high levels of proteases - use fresh inhibitors
May need filtering to remove debris
Consider shorter incubation times to prevent degradation

Adipose Tissue

Very high lipid content - requires special handling
May need lipid removal steps before electrophoresis
Consider using specialized buffers
May need multiple centrifugation steps

Troubleshooting

Low Protein Yield

Possible causes:

Incomplete homogenization (use more vigorous method or extend time)
Insufficient buffer volume (increase buffer-to-tissue ratio)
Proteins lost in pellet (check centrifugation conditions)
Protein degradation (use fresh inhibitors, work faster)

Cloudy or Viscous Supernatant

Solutions:

Filter through 0.45 μm syringe filter
Perform additional centrifugation at higher speed
Dilute sample before filtering
For lipid-rich tissues, consider lipid removal steps

Protein Degradation

Prevention:

Keep tissue frozen until processing
Use fresh protease inhibitors
Work quickly and keep everything cold
Store extracts at -80°C immediately after preparation
For protease-rich tissues (e.g., liver), use stronger inhibitors

High Background in Western Blot