Shreffler: SDS-PAGE

Overview

From wikipedia: "SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a technique widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility (a function of the length of a polypeptide chain and its charge). In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis."

We use the Invitrogen NuPAGE system.

Materials

• Appropriate gelbox/voltage source
• NuPAGE 4-12% BIS-TRIS Gel (Invitrogen NP0322BOX)
• NuPAGE MES SDS Running Buffer (20x) (Invitrogen NP0002)
• NuPAGE SimplyBlue SafeStain (Invitrogen LC6060)
• NuPAGE LDS Sample Buffer (4X) (Invitrogen NP0007)
• NuPAGE Antioxidant (Invitrogen NP0005)
• NuPAGE Reducing Agent (Invitrogen NP0004)

Procedure

Preparing Samples

1. Make a 20 µl loading solution for each protein consisting of:
   • 5 µL LDS Sample Buffer (4X)
   • 2 µL Reducing Agent
   • Volume containing 10 µg protein (e.g. if protein stock solution is 1 µg/µl, add 10 µl)
   • Bring up to 20 µl total with PBS.
2. Heat samples on heating block (or water bath) 70°C for 10 mins (protein denaturing and SDS binding).

Discussion

discuss this protocol

Contact

• Who has experience with this protocol?
• Email Alex Ma through OpenWetWare