NuPAGE electrophoresis/Hybrid staining-PDF

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 the lid of a 1000μL pipette tip box.
• Orbital shaker
• Kimwipes

Procedure

1. Add 100mL deionized water to the staining tray.
2. Microwave staining tray loosely covered for 30 secs on high.
3. Shake the staining tray for 1 min on an 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 the staining tray for 5 mins on an orbital shaker at room temperature.
   • You should now be able to see most of the bands on the gel.
9. Place the staining tray with gel inside a ziploc bag to limit evaporation.
10. Incubate overnight on an 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 the staining tray for 2-3 hours on an orbital shaker at room temperature.
15. Take a 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 a salt solution for several weeks.