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SDS-PAGE
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is an electrophoresis method that allows protein separation based on relative molecular mass.
The basic principle is that proteins of different mass migrate towards the anode with varied resistance, thus allowing proteins to be separated – for example, larger proteins move at a slower rate than smaller polypeptides. To ensure that mass is the only variant, SDS is used as a surfactant that binds polypeptide chains in proportion to their relative molecular mass; it covers proteins' intrinsic charge, conferring them with similar charge-to-mass ratios. If proteins of known mass are run alongside unknown samples, the mass of the unidentified proteins can be estimated and further characterised.
See below for a general SDS-PAGE Protocol.
A. Reagents and Preparation
Reagents
- RIPA Buffer (Radio Immuno Precipitation Assay Buffer)
- RIPA Buffer (Radio Immuno Precipitation Assay Buffer)
- 50 mM Tris, pH 8.0
- 150 mM NaCl
- 1% NP-40
- 0.5% Sodium Deoxycholate
- 0.1% SDS (Sodium dodecyl sulphate)
- Protease Inhibitors
- 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
- Gel (NB. Gel % is dependent on protein size)
- Protein Marker or Ladder
- Loading Control
- Positive and Negative Controls
- Samples
Sample Preparation
- 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. Note: For example, with a BCA test. Leftover serum may affect BCA values.
B. Protocol
- Assemble electrophoresis chamber, ensuring that the running buffer covers all wells.
- Vortex sample with an equal volume of 2X Protein Loading Buffer, and heat to 95°C for 5 mins.
- Vortex again and cool to room temperature before loading.
- Load ~20-40 μg of protein per well.
- Run at 150V for approximately 1hr. Note: Monitor bromophenol blue front. Transfer times depend on protein size.