SDS-PAGE Electrophoresis: Complete Guide
SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) is the foundation of western blotting. This comprehensive guide covers everything you need to know about preparing and running SDS-PAGE gels for optimal protein separation and successful western blot results.
Overview
SDS-PAGE electrophoresis separates proteins based on their molecular weight. The technique uses a polyacrylamide gel matrix and an electric field to drive protein migration. SDS (sodium dodecyl sulfate) denatures proteins and imparts a uniform negative charge, allowing separation based solely on size.
The process involves several key steps:
- Gel preparation - Casting resolving and stacking gels
- Sample loading - Preparing and loading protein samples
- Electrophoresis - Running the gel under appropriate conditions
- Visualization - Staining or transferring for western blot
Proper execution of each step is critical for achieving sharp, well-resolved protein bands that are essential for successful western blotting.
Principle and Mechanism
SDS-PAGE works through several key mechanisms:
Protein Denaturation
SDS is an anionic detergent that binds to proteins at a ratio of approximately 1.4 g SDS per gram of protein. This binding:
- Unfolds proteins into linear chains
- Imparts a uniform negative charge proportional to molecular weight
- Eliminates charge differences between proteins
- Allows separation based solely on size
Gel Matrix
Polyacrylamide gel acts as a molecular sieve:
- Smaller proteins migrate faster through the gel pores
- Larger proteins are more restricted and migrate slower
- Gel percentage determines pore size and separation range
- Stacking gel concentrates proteins before entering resolving gel
Electrophoresis
Electric field drives protein migration:
- Proteins migrate toward the positive electrode (anode)
- Migration rate depends on protein size and gel percentage
- Voltage and running time must be optimized for each application
- Temperature control prevents protein denaturation and gel distortion
Gel Preparation
SDS-PAGE gels consist of two layers: a resolving gel (separation gel) and astacking gel. The resolving gel percentage determines the separation range, while the stacking gel concentrates proteins into a sharp band before separation.
Resolving Gel
The resolving gel is where actual protein separation occurs. Typical percentages:
- 6-8%: For proteins >100 kDa (large proteins)
- 10%: For proteins 30-100 kDa (most common, versatile)
- 12%: For proteins 15-60 kDa
- 15%: For proteins 10-40 kDa
- 18-20%: For proteins <20 kDa (small proteins)
Stacking Gel
The stacking gel (typically 4-5%) concentrates all proteins into a sharp band before they enter the resolving gel. This ensures:
- All proteins start separation at the same point
- Sharp, well-defined bands
- Better resolution and reproducibility
Running Conditions
Proper electrophoresis conditions are essential for optimal protein separation. Key factors include voltage, running time, temperature, and buffer composition.
Standard Conditions
- Voltage: 80-120V constant voltage
- Time: Until dye front reaches 0.5-1 cm from bottom (typically 1-2 hours)
- Notes: Most commonly used, provides good resolution for most proteins
Large Proteins (>100 kDa)
- Voltage: 60-80V constant voltage
- Time: 2-3 hours or until dye front reaches bottom
- Notes: Lower voltage prevents overheating and improves transfer of large proteins
Rapid Separation
- Voltage: 150-200V constant voltage
- Time: 30-60 minutes
- Notes: May cause heating - use cooling system or run in cold room
Gel Percentage Selection Guide
Choosing the correct gel percentage is crucial for optimal protein separation. The gel percentage determines the pore size, which affects how proteins migrate through the gel.
Selection Guidelines
Important Tips
- When in doubt, start with 10% gel - it works for most proteins
- For proteins spanning a wide size range, consider gradient gels
- Very large proteins may require special handling or lower percentage gels
- Small proteins may run off the gel if percentage is too low
Common Issues and Troubleshooting
Poor Resolution
If bands are smeared or poorly resolved, check gel percentage, ensure complete polymerization, use fresh reagents, and optimize running conditions.
Uneven Migration
Uneven dye front or distorted lanes may indicate buffer issues, uneven gel thickness, or temperature fluctuations. Check buffer levels and ensure consistent conditions.
Gel Overheating
Overheating can cause protein denaturation and gel distortion. Reduce voltage, use cooling system, or run in a cold room.
Tips for Better Results
Gel Preparation
- Use fresh APS and TEMED for gel polymerization
- Degas gel solution before adding APS to prevent bubbles
- Allow gel to polymerize completely before use
- Use appropriate gel percentage for your protein size
Running Conditions
- Maintain consistent temperature during electrophoresis
- Ensure buffer levels are adequate
- Use fresh running buffer
- Avoid overloading samples
- Load equal volumes when possible