Composite agarose-polyacrylamide gel electrophoresis (Composite AgPAGE)

Composite agarose-PAGE gel

Materials and equipments:

1. Agarose (SeaKem® HGT(P) Agarose; Catalog No:50050; Lonza).

2. Acrylamide (30% solution, acrylamide/bis 37.5:1; Catalog No:1610158; Bio-Rad).

3. Ammonium persulfate (APS, 40% solution in mpH2O, stored at −20 °C; Bio-Rad).

4. TEMED (Bio-Rad).

5. 5x Tris–HCl buffer (1.875 M, pH 8.1).

6. Glycerol (50% solution).

7. Peristaltic pump (for example, GE Healthcare P-1 pump).

8. Large and small glass plates (1.5-mm), two 0.75-mm combs.

9. Gradient mixer and peristaltic pump (SG Gradient Makers and P-1 pump; GE Healthcare or equivalent).

10. Oven, 60 °C.

11. Gel electrophoresis equipment (Bio-Rad).

12. Running buffer (192 mM boric acid, 1 mM EDTA, 0.1%SDS, pH 7.6 (set with Tris base)).

Methods:

1.Prepare lower and upper gel solution

• Mix the solution well.  
• Boil in microwave for ~1 minute hi power. After first 10 sections stop/shake.    
• Repeat every 5 - 6 seconds until agarose completely dissolved/melted.  
• Place in incubator (50 – 60 °C) wait 30 minutes to let cool to ambient incubator temperature. 

2. Make composite agarose-PAGE gel

• Add 5.1 mL Upper Gel solution with preheated pipette to the upper gel chamber.      
• Open up central channel to let ~1 ml flow through to remove air bubble within the channel. Immediate aspirate the flow through and return it to Upper chamber. 
• Add 6.4 mL Preheated  (30 %) Bis/Acrylamide to Lower Gel Solution. Mix well by swirling. 
• Add 5 mL of this solution to the Lower gel chamber  
• Add 4 uL 10% APS to both Upper and lower chambers.          
• Add 2 uL of TEMED to Upper chamber and Mix with by pipetting up and down times (1 mL setting) with preheated pipette tips.           
• Add 2 uL of TEMED to Lower chamber and mix well as above.            
• Open right valve, immediately start peristaltic pump, then immediately open central channel. 
  • Get Preheated gel in position and position tubing in centre of gel in between plates        
• Fill to top. Two combs (0.75 mm) are put into the gel which is polymerized at RT for at least 6–7 h, or after 1–2 h at RT followed by 4 °C overnight. The gels can be stored (with combs) at 4 °C wrapped in plastic with wet papers for at least a week.
• If the gel around the combs has shrunk, the gel can be reconstituted with the upper gel solution that has been reheated in the microwave oven.

Category