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A. Sample preparation
- 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
- Remove media.
- Wash cells twice to remove residual media using PBS.
- 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.
- 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)
- 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.)
- Transfer supernatant containing the soluble protein to a new tube at 4°C.
- Quantify the protein in the samples. (NB. For example, with a BCA test. Leftover serum may affect BCA values.)
B. Electrophoresis (SDS-PAGE)
- 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
- Assemble electrophoresis chamber, ensuring running buffer covers all wells.
- Vortex sample with an equal volume of 2X Protein Loading Buffer and heat to 95°C for 5 min.
- Vortex again and cool to room temperature before loading.
- Load ~20-40 μg of protein per well.
- Run at 150V for approximately 1h. (NB. Or monitor bromophenol blue front. Transfer times depend on protein size.)