Exercise 9.8 - SDS Gel Electrophoresis of Tubulin\MAPs
Figure 9.3 SDS-PAGE of tubulin and associated proteins
- 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
- 10% (w/v) Ammonium persulphate
- 50% and 7% (w/v) TCA (trichloroacetic acid)
- 0.1% (w/v) Coomassie brilliant blue in 50% TCA
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- 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.
- 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.
- Electrophoresis is carried out at 3 mA per gel until the
bromophenol blue dye reaches the bottom of the tube
(approximately 7 hours).
- Fix the gels overnight in 50% TCA.
- Stain with 0.1% Coomassie brilliant blue (fresh in 50% TCA)
for 1 hour at 37° C.
- Diffusion-destain with repeated washing in 7% TCA.
- Scan the gels or photographically analyze them.
- Determine the molecular weights of the proteins (tubulin and
Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org