LEVEL II

Figure 9.3 SDS-PAGE of tubulin and associated proteins

Materials

• Stock Acrylamide: (30%T:0.8%C)
  
  • 30% by weight of acrylamide
    
  • 0.8% by weight of N,N'-bis-methylene acrylamide
    
• Separation Gel (Final Concentrations)
  
  • 10% acrylamide (1:3 dilution of stock)
    
  • 0.375 M Tris-HCl (pH 8.8)
    
  • 0.1 % SDS
    
• Stacking Gel (Final Concentrations)
  
  • 3 % acrylamide (1:10 dilution of stock)
    
  • 0.125 M Tris-HCl (pH 6.8)
    
  • 0.1% SDS
    
• Electrode Buffer
  
  • 0.025 M Tris
    
  • 0.192 M Glycine
    
  • 0.1% SDS
    
  • Adjust pH to 8.3
    
• 0.2-0.3 ml samples of brain extract, and containing
  
  • 200 micrograms protein
    
  • 0.0625 M Tris-HCl (pH 6.8)
    
  • 2% SDS
    
  • 10% glycerol
    
  • 5% -mercaptoethanol
    
  • 0.001 % bromophenol blue
    
• TEMED
  
• 10% (w/v) Ammonium persulphate
  
• 50% and 7% (w/v) TCA (trichloroacetic acid)
  
• 0.1% (w/v) Coomassie brilliant blue in 50% TCA

Procedure

1. Prepare 15 cm glass tubes with 6 mm internal diameter. Polymerize a 10 cm separation gel and a 1 cm stacking gel within the tubes by the addition of TEMED and ammonium persulfate as directed in Chapter Four.

2. Assemble all the equipment and place 200 µl of sample in one tube. Set up a second gel containing 200 µl of protein molecular weight standards.

3. Electrophoresis is carried out at 3 mA per gel until the bromophenol blue dye reaches the bottom of the tube (approximately 7 hours).

4. Fix the gels overnight in 50% TCA.

5. Stain with 0.1% Coomassie brilliant blue (fresh in 50% TCA) for 1 hour at 37° C.

6. Diffusion-destain with repeated washing in 7% TCA.

7. Scan the gels or photographically analyze them.

8. Determine the molecular weights of the proteins (tubulin and MAP's).