Knight:NuPAGE electrophoresis

In progress! For a better version of this protocol, see Sauer:bis-Tris SDS-PAGE, the very best.

Purpose

To run a denaturing protein gel. Note that a preferable version of this protocol is available at Sauer:bis-Tris SDS-PAGE, the very best (recommended by Kathleen). But since we seem to already have the Invitrogen kit contents in lab, this protocol describes use of the Invitrogen system.

Materials

• NuPAGE Novex 10% Bis-Tris Gel 1.0 mm, 10 well
• XCell SureLock Mini-Cell electrophoresis apparatus
• 20X MES Running Buffer
• NuPAGE LDS Sample Buffer (4X)
• NuPAGE Reducing Agent (10X)
• NuPAGE Antioxidant

Procedure

Sample preparation

Wells can accommodate 25μL loading volume.

1. Each sample should contain
   • 13 μL protein sample (max 0.5μg per band)
   • 5 μL NuPAGE LDS Sample Buffer
   • 2 μL NuPAGE Reducing Agent
2. Heat samples at 70°C for 10 mins.

Running buffer preparation

1. Prepare 1000mL 1X NuPAGE SDS running buffer
   • 50mL 20X MES Running Buffer
   • 950mL deionized water
2. Mix well.
3. Set aside 200mL buffer for use in Upper (Inner) buffer chamber).
   • Add 500μL NuPAGE Antioxidant immediately before use.
   • Mix well.

References

1. NuPAGE Technical Guide