Knight:NuPAGE electrophoresis/Hybrid staining

Overview

A fast protocol for visualizing bands on a polyacrylamide gel via coomassie-like staining.

Materials

• Deionized water
• Invitrogen SimplyBlue SafeStain
• Microwave
• Plastic tray
  • Use lid of a 1000μL pipette tip box.
• Orbital shaker
• Kimwipes

Procedure

1. Add 100mL deionized water in staining tray.
2. Microwave staining tray loosely covered for 30 secs on high.
3. Shake staining tray for 1 min on orbital shaker at room temperature.
4. Discard water.
5. Repeat steps 1-4 two more times.
6. Add 20mL SimplyBlue SafeStain (enough to just cover the gel).
7. Microwave staining tray loosely covered for 20 secs on high.
8. Shake staining tray for 5 mins on orbital shaker at room temperature.
   • You should now be able to see most of the bands on the gel.
9. Place staining tray with gel inside a ziploc bag to limit evaporation.
10. Incubate overnight on orbital shaker at room temperature.
    • Overnight incubation will yield better sensitivity due to darker staining.
11. Discard SimplyBlue SafeStain.
12. Add 100mL deionized water.
13. Add 2 kimwipes.
14. Shake staining tray for 2-3 hours on orbital shaker at room temperature.
15. Take gel picture.
16. Add 20mL 20% NaCl for at least 5 mins.
    • Only do this step if you aren't planning on drying the gel! Otherwise, when the gel dries, you will see a white salt precipitate.
17. Gel can be stored in salt solution for several weeks.