User:Douglas M. Fox/Notebook/AU CHEM-571 F2011 Lab Support/2014/10/14

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SDS-PAGE #2

  • Mix 10 μL 0.6 g/L lysozyme with 10μL SDS-PAGE sample buffer in 1.5 mL centrifuge vial
  • Mix 10 μL 0.12 g/L lysozyme with 10μL SDS-PAGE sample buffer in 1.5 mL centrifuge vial
  • Mix 10 μL 30:1 Au/lysozyme colloid with 10μL SDS-PAGE sample buffer in 1.5 mL centrifuge vial
  • Mix 10 μL 0.12 g/L unknown protein with 10 μL SDS-PAGE sample buffer in 1.5 mL centrifuge vial
  • Place in heating block (set at 90 °C) for 5 minutes
  • Store in refrigerator overnight
  • One group prepare additional 1 L of running buffer from 10X concentrate


Note: Ideal concentration for pure protein is 0.5 - 4.0 μg (40 - 60 μg for crude samples).
Using 10 μL of 0.12 g/L will load 1.2 μg protein into the well if entire 20 μL is used.

Protein Dialysis

  • Continue dialysis from previous tasklist

Buffer Preparation

  • Prepare 30 - 40 mL 66 mM potassium phosphate buffer, pH 6.24
    • Use HPLC water
    • 8.98 mg/mL KH2PO4 should produce pH 4 - 4.1
    • Add 1 M KOH to adjust pH to 6.24. To avoid this step a mixture of monobasic and dibasic potassium phosphate was used.
    • Store