Shreffler: SDS-PAGE

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

Revision as of 13:34, 22 March 2012

Contents

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.
  1. Heat samples on heating block (or water bath) 70°C for 10 mins (protein denaturing and SDS binding).

Running Gel

Discussion

discuss this protocol

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