How to Make an SDS-PAGE Gel: Step-by-Step Guide

SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is a cornerstone technique in molecular biology and biochemistry for separating proteins based on their size. In this post, we’ll walk you through how to prepare an SDS gel, including tips for separation and stacking gels, using a typical 10% SDS-PAGE gel recipe.

Whether you’re a beginner or an experienced researcher, this step-by-step guide will help you make reliable gels for your experiments.

Safety notes:

• Acrylamide is neurotoxic → always wear gloves & goggles.

• Use fresh APS (unstable in solution).

• Add TEMED last → starts polymerization immediately.

Resolving Gel (for 100 ml, ×10)

Reagent	7 %	8 %	10 %	12 %	15 %
H₂O	3.8 ml	3.4 ml	2.8 ml	2.1 ml	1.1 ml
30 % Acrylamide	2.3 ml	2.7 ml	3.3 ml	4.0 ml	5.0 ml
1 M Tris-HCl pH 8.8	3.7 ml	3.7 ml	3.7 ml	3.7 ml	3.7 ml
10 % SDS	100 µl	100 µl	100 µl	100 µl	100 µl

Polymerization (per 10 ml):

• Add freshly: 80 µl ammonium persulfate (APS) + 10 µl TEMED

• Pipette ~5 ml into a 1 mm gel cassette

• Overlay with isopropanol to avoid air bubbles

• Let polymerize ~20 min

• Discard isopropanol

Stacking Gel (for 3 ml, usually 5 %)

Reagent	4 %	5 %	6 %	7 %
H₂O	2.200 ml	2.140 ml	2.040 ml	1.893 ml
30 % Acrylamide	0.390 ml	0.488 ml	0.584 ml	0.732 ml
1 M Tris-HCl pH 6.8	0.375 ml	0.375 ml	0.375 ml	0.375 ml
10 % SDS	30 µl	30 µl	30 µl	30 µl

Polymerization (per 3 ml):

• Add freshly: 15 µl APS + 3 µl TEMED

• Pipette ~1 ml into a 1 mm gel cassette

• Insert comb carefully (avoid bubbles, wear safety glasses)

• Let polymerize ~20 min

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Materials You’ll Need

• Glass plates and casting stand

• Acrylamide solution

• Tris buffer (pH 8.8 for separation gel, pH 6.8 for stacking gel)

• SDS (Sodium Dodecyl Sulfate)

• TEMED (Tetramethylethylenediamine)

• APS (Ammonium Persulfate)

• Water or isopropanol

• Phenol red (optional, for visualizing stacking gel)

• Pipettes and vortex mixer

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Step 1: Assemble the Gel Apparatus

1. Stack two glass plates together and place them in the casting stand.

2. Ensure the bottom of the plates is parallel to prevent leaks.

3. Secure the plates firmly. You can prepare multiple gels at once if desired—up to 10 channels is possible with practice.

4. Add water to check for leaks; let it stand a few minutes. If no water escapes, the setup is ready.

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Step 2: Prepare the Separation Gel

The separation (resolving) gel separates proteins based on size.

1. Combine the following in a clean container:

   • Water

   • Acrylamide solution

   • Tris buffer pH 8.8

   • SDS

2. Just before pouring, add APS and TEMED to initiate polymerization.

3. Vortex the solution thoroughly to mix all components.

4. Using a pipette, pour the separation gel solution between the glass plates.

5. Carefully overlay with isopropanol (or water) to prevent contact with air, which can interfere with polymerization.

6. Allow the gel to polymerize completely (usually 20–30 minutes).

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Step 3: Prepare the Stacking Gel

The stacking gel concentrates proteins into sharp bands before entering the separation gel.

1. Mix:

   • Water

   • Acrylamide solution

   • Tris buffer pH 6.8

   • SDS

   • Phenol red (optional, for visual distinction)

2. Add APS and TEMED, then vortex gently.

3. Remove the isopropanol from the top of the separation gel.

4. Pour the stacking gel on top of the polymerized separation gel.

5. Insert the comb carefully to create wells for loading protein samples.

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Step 4: Tips for Successful SDS-PAGE Gel Preparation

• Work quickly after adding TEMED and APS, as polymerization begins immediately.

• Use phenol red in the stacking gel to easily distinguish it from the separation gel.

• Ensure the glass plates are clean and leak-proof.

• Avoid bubbles while pouring gels—they can interfere with protein migration.

• Multiple gels can be prepared simultaneously if your casting stand allows it.

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Step 5: Ready to Run

Once both gels have polymerized and the comb is in place, your SDS-PAGE gel is ready for protein loading and electrophoresis. This process ensures clear, reproducible separation of proteins by size.

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Final Thoughts

Preparing an SDS-PAGE gel may seem daunting at first, but with careful assembly, accurate solution preparation, and proper polymerization techniques, you can create high-quality gels for your experiments. Following these step-by-step instructions ensures reliable protein separation every time.

✅ Pro tip: Always check for leaks before adding polymerizing gel solutions and work quickly once APS and TEMED are added.