Preparing Acrylamide Gels for Electrophoresis: Protocols and Best Practices

Preparing Acrylamide Gels for Electrophoresis

Acrylamide gel electrophoresis is a widely used technique in molecular biology for separating proteins and nucleic acids based on size and charge. Proper preparation of acrylamide gels is crucial for obtaining accurate and reproducible results in protein analysis, DNA sequencing, and RNA studies. Understanding the right protocols, gel composition, and polymerization conditions ensures optimal resolution and minimizes experimental errors. 

Laboratory setup showing the pouring of acrylamide solution into a gel casting apparatus for electrophoresis

Acrylamide gels are prepared by polymerizing acrylamide and bisacrylamide with catalysts to form a matrix that separates proteins or nucleic acids based on size during electrophoresis.

Setting Up the Work Area

A clean and organized workspace is critical for accurate gel preparation. Start by assembling the necessary tools:

  1. White absorbent paper: Protects surfaces from spills.
  2. Spill catch tray: Minimizes contamination risks.
  3. Plate support: Use a sturdy styrofoam block to hold plates steady.

This setup prevents cross-contamination and simplifies cleanup, ensuring a smooth workflow.

Cleaning and Preparing Glass Plates

Proper treatment of glass plates is essential for uniform gel formation:

  1. New Plates:
    • Soak in 2M sodium hydroxide for at least one hour to remove residues.
    • Treat one side of the shorter plate with Rain-X to prevent sticking.
  2. Routine Maintenance:
    • After 10 uses, clean the long plate with 2N NaOH and reapply Rain-X to the shorter plate.

Cleaning Steps:

  • Wash long plates in the sink using Alconox or a similar detergent.
  • Rinse thoroughly with distilled water, ensuring all detergent is removed.
  • Dry the non-gel side with paper towels and the gel side with Kimwipes.
  • Apply 95% ethanol to remove any remaining residues. Repeat for the short plate.

This meticulous cleaning prevents imperfections in the gel and ensures even polymerization.

Assembling the Gel Mold

  1. Place spacers along the edges of the long plate.
  2. Position the short plate on top, aligning it precisely to avoid gaps.
  3. Secure the plates with binder clips, ensuring even pressure.
  4. Add a third spacer at the bottom for structural stability, securing it with additional clips.

Proper assembly ensures uniform gel thickness and prevents leaks during pouring.

Preparing the Gel Solution

Ingredients for a 60 ml Gel:

  • 5× TBE buffer: 12 ml
  • Distilled water (dH₂O): 13 ml
  • Urea: 27 g
  • 40% Acrylamide Stock: 9 ml (preparation: 38 g acrylamide + 2 g bis-acrylamide per 100 ml)

Final Gel Composition:

  • Acrylamide: 5.7%
  • Bis-Acrylamide: 0.3%
  • Urea: 45% (7.49 M)
  • Tris: 0.089 M
  • Boric Acid: 0.089 M
  • EDTA: 0.002 M

Steps to Prepare Gel Solution:

  1. Mix TBE buffer and water in a beaker.
  2. Warm the mixture on a hot plate and dissolve urea completely.
  3. Remove from heat and adjust the volume to 51 ml with dH₂O.
  4. Filter the solution through a 0.45 μm membrane filter into an acrylamide squirt bottle.
  5. Add acrylamide stock, swirling gently to mix.

Add TEMED (30 μl) and 10% APS (320 μl) just before pouring to initiate polymerization.

Pouring and Setting the Gel

  1. Pour the prepared solution carefully between the glass plates.
  2. Use a bubble grabber to remove trapped air bubbles.
  3. Insert combs at a depth of 4 mm and cover with extra acrylamide solution.
  4. Tilt the assembly at a 15° angle and allow polymerization for 1–1.5 hours.
  5. Discard excess solution into a designated waste bottle.

Cover the gel with moistened Kimwipes and plastic film if storing overnight to prevent drying.

Running the Gel

  1. Assemble the gel in the electrophoresis apparatus and tighten clamps firmly.
  2. Prepare 1× TBE buffer (160 ml 5× TBE diluted to 800 ml with water).
  3. Fill the top reservoir to cover the short plate and rinse the wells with a syringe.
  4. Pour remaining TBE into the bottom reservoir, ensuring no bubbles are trapped.

Pre-Run:

Run the gel at 60 watts for 30 minutes to equilibrate. Clean the wells with a syringe before loading samples.

Sample Preparation and Gel Electrophoresis

  1. Denature samples at 90°C for 3 minutes and cool on ice.
  2. Load samples carefully to avoid damaging the gel surface.
  3. Run the gel at 60 watts for 2 hours, monitoring progress to ensure proper separation.

Disassembling and Drying the Gel

  1. Turn off the power and drain the upper reservoir.
  2. Cool the plates on ice for 2–3 minutes.
  3. Separate the plates, ensuring the gel adheres to the long plate.
  4. Transfer the gel to Whatman 3MM paper, smoothing out air bubbles.
  5. Dry the gel at 80°C for 50 minutes under vacuum.

Imaging and Documentation

Place the dried gel against X-ray film in a cassette for 36–72 hours. This step allows visualization of separated molecules using autoradiography.

Waste Disposal and Cleanup

Dispose of liquid waste in radioactive waste bottles, ensuring compliance with safety regulations. Clean all equipment thoroughly to prevent contamination.

Troubleshooting Tips

  • Uneven Gel Polymerization: Ensure TEMED and APS are fresh and mixed thoroughly.
  • Bubbles in Gel: Use a bubble grabber and pour slowly to minimize air entrapment.
  • Poor Sample Loading: Align combs properly and rinse wells thoroughly before loading.

Preparing acrylamide gels requires precision and attention to detail. By following these comprehensive steps, researchers can achieve consistent results, minimizing errors and maximizing the efficiency of their electrophoresis experiments. 

Mastering the preparation of acrylamide gels is essential for achieving precise and consistent results in electrophoresis experiments. By following standardized protocols, optimizing gel composition, and maintaining proper polymerization conditions, researchers can enhance the accuracy and reproducibility of their analyses. As electrophoresis continues to play a critical role in molecular biology, refining gel preparation techniques ensures better separation efficiency and reliable data interpretation. With these best practices, scientists can confidently conduct protein and nucleic acid analysis, driving advancements in genetics, biochemistry, and biomedical research.