SDS-Page

A. Sample preparation

Reagents

  • RIPA Buffer (Radio Immuno Precipitation Assay Buffer) contains:
    • 50 mM Tris, pH 8.0
    • 150 mM NaCl
    • 1% NP-40
    • 0.5% Sodium Deoxycholate
    • 0.1% SDS (Sodium dodecyl sulphate)
  • Protease Inhibitors
  1. Remove media.
  2. Wash cells twice to remove residual media using PBS.
  3. Remove PBS and add appropriate volume of RIPA Buffer plus protease inhibitors (1 ml per 0.5 to 5 x 107 cells). Incubate at 4°C for 5 min.
  4. Dislodge cells using a cell scraper and transfer to a tube at 4°C. (NB. If needed, store at -70°C at this point. Storing after denaturation may cause aggregation)
  5. Spin the lysate at 8000 x g for 10 min at 4°C to pellet the cell debris. (NB. Before centrifugation, remove nucleic acid aggregates which may form at the top using a pipette.)
  6. Transfer supernatant containing the soluble protein to a new tube at 4°C.
  7. Quantify the protein in the samples. (NB. For example, with a BCA test. Leftover serum may affect BCA values.)

B. Electrophoresis (SDS-PAGE)

Reagents

  • Gel (NB. Gel % is dependent on protein size)
  • 2X Protein Loading Buffer
    • 125 mM Tris HCl
    • 4% SDS
    • 10% β-mercaptoehtanol
    • 20% glycerol
    • 0.004% bromophenol blue
    • pH ~6.8
  • Running Buffer
    • 25 mM Tris base
    • 190 mM Glycine
    • 0.1% SDS
    • pH ~8.3
  • Protein Marker or Ladder
  • Loading Control
  • Positive and Negative Controls
  • Samples
  1. Assemble electrophoresis chamber, ensuring running buffer covers all wells.
  2. Vortex sample with an equal volume of 2X Protein Loading Buffer and heat to 95°C for 5 min.
  3. Vortex again and cool to room temperature before loading.
  4. Load ~20-40 μg of protein per well.
  5. Run at 150V for approximately 1h. (NB. Or monitor bromophenol blue front. Transfer times depend on protein size.)