Shreffler: SDS-PAGE

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

Running Gel

  1. Make 1X
  2. Remove precast 4-12% Bis-Tris gels from packages, carefully and over a sink (pouch contains liquid). Pull off white stripe at bottom and carefully remove plastic comb from gel. Gently wash cassette and (esp. wells) under DI water (can apply gentle centrifugal force to eject residual liquid from wells so that they can be washed).
  3. Lower buffer core into cell and insert gel cassettes on both sides, such that the shorter well side faces inward (if only running one gel, use a dummy cassette). Insert tension wedge and lock in place.

Fill the buffer core with 200 mL 1X RB. Make sure this section does NOT leak before adding liquid to the outside. Carefully remove the comb and pipette out any air bubbles. Then, fill outer part of case with 600 mL of RB. Fill the wells with samples: Add 10 μL MW markers to well #1. Fill remaining 11 wells with 10 μL of prepared protein sample solution. Fill each well slowly. Try to avoid bubbles in wells that may cause overflow into other wells When working with 2 gel plates, make sure to label the outside of the container. Match the + and -electrode ends (same colors) Turn on (be sure Range Select is OFF). Set Range to 200V/400mA, then hit Start. Run for 35 min. When done, then turn off. Gel stain: Take gel cassette out of Invitrogen gel box. Fill microwave safe glass container with MilliQ water enough as to immerse gel. Crack open gel cassette and place gel in microwave safe glass container. Microwave gel for two minutes or until water boils. Place container on shaker until cool. Then decant the water. Rinse one more time with MilliQ water. Add Denville Blue Protein Stain to gel container and immerse gel. Microwave gel for two minutes or until protein stain solution boils and place on shaker until cool. Decant gel stain into waste container, and rinse gel with MilliQ water. Add MilliQ water to immerse gel and microwave for two minutes and then shake until cool. Carefully place gel into plastic gel box filled with water.

Discussion

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