Is There a Protocol for Blue Native PAGE and How Should the Gel Be Prepared?

The following describes a basic procedure for preparing BN-PAGE gels. The specific parameters (e.g., concentration, pH) should be optimized according to the characteristics of the sample and the experimental objectives.

Gel Solution Formulation

1. Separating Gel (12%)

• 30% acrylamide solution: 12 mL
• 1.5 M Tris-HCl, pH 8.8: 12.5 mL
• Deionized water: 9.4 mL
• 10% APS: 500 µL
• TEMED: 50 µL

2. Stacking Gel (4%)

• 30% acrylamide solution: 1.33 mL
• 1.0 M Tris-HCl, pH 6.8: 2.5 mL
• Deionized water: 6.07 mL
• 10% APS: 100 µL
• TEMED: 10 µL

Gel Casting Procedure

1. Preparation of Separating Gel Solution

Combine all components according to the separating gel formulation. Add APS and TEMED last, mix thoroughly, and proceed immediately to casting.

2. Casting the Separating Gel

Introduce the separating gel solution between the pre-cleaned and assembled glass plates, leaving adequate space for the stacking gel. Overlay the gel surface with anhydrous ethanol or deionized water to prevent oxygen inhibition of polymerization. Allow the gel to polymerize completely.

3. Preparation of Stacking Gel Solution

Once the separating gel has polymerized, remove the overlaying ethanol or water. Prepare the stacking gel solution according to the formulation above, adding APS and TEMED last.

4. Casting the Stacking Gel

Overlay the stacking gel solution onto the polymerized separating gel, and immediately insert the comb to form sample wells.

5. Polymerization and Storage

After complete polymerization of the stacking gel, the electrophoresis gel is ready for use, or it may be wrapped in a moistened paper towel and stored at 4°C until required.

MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.

Related Services

SDS-PAGE Based Protein Purity Analysis Service