Porcine brain tubulin preparation
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实验试剂
100g P-11 cellulose phosphate fibrous cation exchanger (Whatman Inc, Clifton NJ) 6L 0.1M HCl
~10 L 1x column buffer 10 M NaOH
300 ml Homogenization buffer (freshly prepared)
1L PMG Buffer 100 mM MgGTP 1M dithiothreitol (DTT)
实验设备
Waring Blender Temperature controlled ultracentrifuge with the equivalent of a Beckman 50.2Ti rotor
At least 24 31.5 ml thick-walled polycarbonate ultracentrifuge tubes with screw caps
30 ml Dounce "A" glass homogenizer
2L scintered glass filter funnel and 2L sidearm Erlenmeyer flask Amicon (Danvers, MA)
Fraction collector UV monitor or Bradford reagent (Biorad)
实验材料
3 fresh (<3 hrs after slaughter) pig brains
实验步骤
DAY 1: Preparation of the phosphocellulose column.
6. Repeat the 0.1 M HCl treatment cycle until the pH of the phosphocellulose slurry is below 3.
7. Rinse the phosphocellulose with 4 L of distilled water by vacuum filtration.
DAY 2: Cycling preparation of MT protein.
5. Homogenize the tissue by blending 5 s on high speed and then 45 s on low speed.
6. This should result in a suspension with the color and consistency of a strawberry milkshake.
12. During this polymerization incubation, warm the ultracentrifuge and 50.2Ti rotor to 25℃.
17. During this time chill the ultracentrifuge and 50.2Ti rotor to 4℃.
21. During the incubation, warm the ultracenrtifuge and 50.2Ti rotor to 25℃.
DAY 3: Phosphocellulose column purification of tubulin from MT protein