Western Blot Hub

SDS-PAGE Troubleshooting: Common Problems & Solutions

Encountering problems with SDS-PAGE electrophoresis? This comprehensive troubleshooting guide covers common issues, their causes, and step-by-step solutions to help you achieve optimal protein separation and successful western blot results.

Poor Resolution

Problem: Smeared or Blurry Bands

Possible Causes:

  • Incorrect gel percentage for protein size
  • Incomplete gel polymerization
  • Old or expired reagents (APS, TEMED)
  • Sample overloaded (too much protein)
  • Improper running conditions (voltage too high or too low)
  • Protein degradation in sample

Solutions:

  • Use appropriate gel percentage (10% for most proteins, adjust for size)
  • Ensure complete polymerization (wait 30-45 min for resolving gel)
  • Use fresh APS and TEMED (prepare APS fresh daily)
  • Reduce sample amount (try 20-30 μg instead of 50 μg)
  • Optimize voltage (80-120V for standard conditions)
  • Check sample preparation - ensure proper denaturation

Problem: Bands Too Close Together

Possible Causes:

  • Gel percentage too high for protein size
  • Insufficient running time
  • Voltage too low

Solutions:

  • Use lower gel percentage (e.g., 8% instead of 12%)
  • Extend running time until dye front reaches bottom
  • Increase voltage slightly (within safe range)

Uneven Migration

Problem: Uneven Dye Front

Possible Causes:

  • Uneven buffer levels in upper and lower chambers
  • Buffer contamination or incorrect pH
  • Uneven gel thickness
  • Temperature fluctuations
  • Gel cassette not properly assembled

Solutions:

  • Ensure equal buffer levels in both chambers
  • Use fresh running buffer with correct pH (8.3)
  • Check gel cassette assembly - ensure even spacing
  • Maintain consistent temperature (use cooling if needed)
  • Verify gel casting was done on level surface

Problem: Distorted Lanes

Possible Causes:

  • Uneven sample loading volumes
  • Sample spilled into adjacent wells
  • Bubbles in wells
  • Gel damage or bubbles in gel matrix

Solutions:

  • Load equal volumes in all wells
  • Load samples carefully and slowly
  • Remove bubbles from wells before loading
  • Check gel for bubbles or damage before use

Gel Preparation Issues

Problem: Gel Won't Polymerize

Possible Causes:

  • Old or expired APS or TEMED
  • Incorrect pH of buffers
  • Too much oxygen in solution (insufficient degassing)
  • Temperature too low
  • Incorrect reagent concentrations

Solutions:

  • Use fresh APS (prepare daily) and TEMED
  • Check buffer pH (8.8 for resolving, 6.8 for stacking)
  • Degas thoroughly (10-15 minutes under vacuum)
  • Work at room temperature (20-25°C)
  • Verify reagent concentrations and volumes

Problem: Bubbles in Gel

Possible Causes:

  • Vortexing after adding TEMED
  • Insufficient degassing
  • Pouring too quickly
  • Air bubbles introduced during pouring

Solutions:

  • Mix gently by swirling after adding TEMED (never vortex)
  • Degas thoroughly before adding polymerization agents
  • Pour slowly and carefully
  • Tap gel cassette gently to remove bubbles after pouring

Problem: Uneven Gel Surface

Possible Causes:

  • Improper overlay technique
  • Uneven pouring
  • Gel cassette not level
  • Incomplete polymerization

Solutions:

  • Use proper overlay technique (isopropanol or water-saturated butanol)
  • Pour evenly across the gel cassette
  • Ensure gel casting stand is level
  • Allow complete polymerization before removing overlay

Overheating Problems

Problem: Gel Overheating

Possible Causes:

  • Voltage too high
  • Insufficient cooling
  • Running in warm environment
  • Buffer conductivity issues

Solutions:

  • Reduce voltage (use 80-100V instead of 150V+)
  • Use cooling system or run in cold room
  • Monitor temperature - should stay below 30°C
  • Use fresh running buffer with correct composition
  • Consider running at lower voltage for longer time

Problem: Protein Denaturation

Possible Causes:

  • Overheating during electrophoresis
  • Insufficient sample denaturation before loading
  • Protein degradation in sample

Solutions:

  • Control temperature during run (use cooling)
  • Heat samples at 95°C for 5 minutes before loading
  • Use fresh samples and proper storage conditions
  • Add protease inhibitors if needed

Band Problems

Problem: No Bands Visible

Possible Causes:

  • Sample not loaded properly
  • Proteins ran off the gel
  • Insufficient protein amount
  • Protein degradation
  • Staining issues (if using Coomassie)

Solutions:

  • Verify samples were loaded correctly
  • Check if proteins are too small (may need higher % gel)
  • Increase protein amount (try 50-100 μg)
  • Check sample preparation and storage
  • Verify staining protocol if using Coomassie

Problem: Multiple Bands for Single Protein

Possible Causes:

  • Protein isoforms or post-translational modifications
  • Incomplete denaturation
  • Protein aggregation
  • Contamination

Solutions:

  • This may be normal for some proteins (check literature)
  • Ensure complete denaturation (heat at 95°C, use reducing agent)
  • Check sample preparation for aggregation
  • Verify sample purity

Prevention Tips

Gel Preparation

  • Always use fresh reagents (APS, TEMED)
  • Degas gel solutions thoroughly
  • Allow complete polymerization before use
  • Use appropriate gel percentage for your protein
  • Work on a level surface

Running Conditions

  • Use fresh running buffer
  • Maintain consistent temperature
  • Monitor voltage and current
  • Ensure adequate buffer levels
  • Load samples carefully and consistently

Sample Preparation

  • Properly denature samples (95°C for 5 min)
  • Use appropriate protein amounts
  • Include molecular weight markers
  • Store samples properly to prevent degradation

Related Protocols

SDS-PAGE Electrophoresis Overview

Complete guide to SDS-PAGE electrophoresis

Gel Preparation Protocol

Detailed guide to preparing SDS-PAGE gels

Running Conditions

Guide to electrophoresis conditions

Western Blot Troubleshooting

Troubleshooting guide for western blotting