Knight:NuPAGE electrophoresis
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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.
- Each sample should contain
- 13 μL protein sample (max 0.5μg per band)
- 5 μL NuPAGE LDS Sample Buffer
- 2 μL NuPAGE Reducing Agent
- Heat samples at 70°C for 10 mins.
Running buffer preparation
- Prepare 1000mL 1X NuPAGE SDS running buffer
- 50mL 20X MES Running Buffer
- 950mL deionized water
- Mix well.
- Set aside 200mL buffer for use in Upper (Inner) buffer chamber).
- Add 500μL NuPAGE Antioxidant immediately before use.
- Mix well.