SDS-PAGE Gel Preparation: Step-by-Step Protocol
Proper gel preparation is essential for successful SDS-PAGE electrophoresis. This detailed protocol provides step-by-step instructions for preparing both resolving and stacking gels, including recipes, calculations, and troubleshooting tips.
Overview
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.
Key components of gel preparation:
- Acrylamide/Bis solution: Forms the gel matrix (typically 29:1 or 37.5:1 ratio)
- Tris buffers: Maintains pH (pH 8.8 for resolving gel, pH 6.8 for stacking gel)
- SDS: Ensures protein denaturation and uniform charge
- APS and TEMED: Polymerization initiators
The gel percentage (acrylamide concentration) determines pore size and affects protein migration. Choose the appropriate percentage based on your target protein size (see Gel Percentage Calculations).
Materials Required
Reagents
- • 30% Acrylamide/Bis solution (29:1 or 37.5:1 ratio)
- • 1.5 M Tris-HCl, pH 8.8 (for resolving gel)
- • 0.5 M Tris-HCl, pH 6.8 (for stacking gel)
- • 10% SDS (sodium dodecyl sulfate)
- • 10% APS (ammonium persulfate) - prepare fresh
- • TEMED (N,N,N',N'-Tetramethylethylenediamine)
- • Deionized water
- • Isopropanol or water-saturated butanol (for overlay)
Equipment
- • Gel casting system (glass plates, spacers, combs)
- • Gel casting stand
- • Vacuum pump (for degassing, optional but recommended)
- • Graduated cylinders or pipettes for measuring volumes
- • Filter paper (for drying gel surface)
Resolving Gel Preparation
The resolving gel is where actual protein separation occurs. The percentage determines the separation range. Here's a detailed protocol for preparing a 10% resolving gel (adjust volumes for different percentages):
Step 1: Calculate Volumes
For a 10% resolving gel (10 mL total volume):
- 3.3 mL 30% acrylamide
- 2.5 mL 1.5 M Tris-HCl pH 8.8
- 0.1 mL 10% SDS
- 4.05 mL deionized water
Important: Degas under vacuum for 10-15 minutes to remove dissolved oxygen, which can interfere with polymerization.
Step 2: Add Polymerization Agents
After degassing, add polymerization initiators:
- 50 μL 10% APS (ammonium persulfate)
- 10 μL TEMED
Mix gently by swirling - do not vortex as this introduces bubbles that can disrupt gel structure.
Pour immediately into gel cassette to about 1 cm below where the comb will sit. Work quickly as polymerization begins immediately after adding TEMED.
Step 3: Overlay
Carefully overlay with isopropanol or water-saturated butanol to:
- Prevent oxygen contact (oxygen inhibits polymerization)
- Create a flat gel surface
- Ensure even polymerization
Allow to polymerize for 30-45 minutes at room temperature. You will see a clear interface when polymerization is complete.
Step 4: Remove Overlay
Once polymerization is complete:
- Pour off the overlay carefully
- Rinse top of gel with deionized water
- Dry the top surface with filter paper
- Be careful not to touch the gel surface
The resolving gel is now ready for stacking gel preparation.
Stacking Gel Preparation
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, resulting in sharp, well-defined bands.
Step 1: Prepare Stacking Gel Solution
For 5% stacking gel (4 mL total volume):
- 0.67 mL 30% acrylamide
- 1.0 mL 0.5 M Tris-HCl pH 6.8
- 0.04 mL 10% SDS
- 2.25 mL deionized water
Degas if time permits, though it's less critical for stacking gel.
Step 2: Add Polymerization Agents
Add polymerization initiators:
- 25 μL 10% APS
- 5 μL TEMED
Mix gently and pour on top of resolving gel immediately. Work quickly as polymerization is rapid.
Step 3: Insert Comb
Quickly insert the comb at an angle to avoid bubbles, then straighten it. This creates the wells for sample loading.
Allow to polymerize for 20-30 minutes. The gel should be ready when you can see a clear line at the comb teeth.
Step 4: Remove Comb
Once polymerization is complete:
- Carefully remove the comb
- Rinse wells with running buffer or deionized water
- Remove any unpolymerized acrylamide
The gel is now ready for sample loading and electrophoresis.
Gel Percentage Calculations
The gel percentage is calculated as the percentage of acrylamide in the total gel volume. Here's how to calculate volumes for different gel percentages:
Formula
For a gel with total volume V and desired percentage P:
The remaining volume is made up of buffer, SDS, water, and polymerization agents.
Gel Percentage Guidelines
- 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)
Important Notes
- Always use fresh 30% acrylamide solution
- Store acrylamide solution protected from light at 4°C
- Check expiration date - old acrylamide may not polymerize properly
- For gradient gels, prepare two solutions with different percentages
Troubleshooting
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
Solutions: Use fresh reagents, check buffer pH, degas thoroughly, work at room temperature.
Bubbles in Gel
Possible causes:
- Vortexing after adding TEMED
- Insufficient degassing
- Pouring too quickly
Solutions: Mix gently by swirling, degas before adding initiators, pour slowly and carefully.
Uneven Gel Surface
Possible causes:
- Improper overlay
- Uneven pouring
- Gel cassette not level
Solutions: Ensure level surface, pour evenly, use proper overlay technique.