Native PAGE

Reagents and Materials

• SDS PAGE Gel apparatus: lid, tank, combos, spacer plates, short plates, casting frame and casting stand

5% Native PAGE Gel (Separating)

• 1.875 mL Acryl/Bis (40%)
• 1.3 mL 0.375M Tris (pH 8.8)
• 50 µL 10% APS
• 5 µL TEMED

4% Native PAGE Gel (Stacking)

• 1.875 mL Acryl/Bis (40%)
• 1.3 mL 0.375M Tris (pH 8.8)
• 30 µL 10% APS
• 3 µL TEMED

Native PAGE Running Buffer

• 25mM Tris
• 192mM Glycine

4x Native PAGE Loading Dye

• 62.5mM Tris-HCl (pH 6.8)
• 25% Glycerol
• 1% Bromophenol Blue

Protocol

1. Put two gel glass pieces together in the correct orientation (should be small space between them) and clip into the gel apparatus
2. Add about 5 mL of separating gel in between the two glass pieces (ensure it does not leak)
3. Add a thin layer of isopropanol on top of the separating gel to form a smooth line
4. Wait for the gel to solidify (~ 20 minutes)
5. Rinse out the isopropanol with water
6. Add about 2 mL of the stacking gel on top of the separating gel and add the gel comb
7. Wait for the gel to solidify (~ 20 minutes)
8. Place the gel into the gel running apparatus and lock it in place. Ensure the side with the larger piece of glass is facing outward. If there is only one gel, add empty glass pieces to the other side
9. Put the gel into the gel box and fill with 1x Native PAGE Running Buffer. Ensure the middle section between the gels is filled with buffer
10. Load samples, DO NOT HEAT SAMPLES
11. Run gel at 100V until samples leave the well and increase the voltage to 150V. Or run the gel at lower voltages (~90V) to get cleaner bands
12. When the gel is done running, fix it with destaining solution (50% water, 40% methanol, 10% acetic acid) for 30 min
13. Stain the gel with Coomassie Brilliant Blue, or 4% HCl and 2% Potassium ferrocyanide for 1h
14. Destain the gel with water or destaining solution