To begin, take the clarified extract of the homogenized porcine brain tissue. Pour the extract into pre-chilled ultracentrifugation tubes, and centrifuge at 75, 000G for 60 minutes at four degrees Celsius. Collect the supernatant, and measure its volume S1.Next, add 10 times MEM buffer to the supernatant, and mix thoroughly.
Add glycerol and GTP to a final concentration of 3.5 molar and 0.1 millimolar, respectively. Pour the suspension into ultracentrifugation tubes, and incubate for 45 minutes at 37 degrees Celsius in a water bath. After that, centrifuge the tubes at 75, 000G for 90 minutes at 37 degrees Celsius.
Then, measure the volume of the supernatant, S2, before discarding it. Now, add an equal amount of ice cold MEM buffer to the pellets. After resuspending the pellets, transfer the suspensions to a graduated cylinder, and determine the total volume.
Then, add GTP to a final concentration of one millimolar. Transfer the suspension into a downs glass homogenizer, and homogenize the solution intermittently every 10 minutes for 45 minutes on ice. Pour the homogenized solution into pre-chilled ultra centrifugation tubes.
Centrifuge at 75, 000G for 60 minutes at four degrees Celsius, and determine the volume of the supernatant, S3.Now, mix the supernatant with glycerol. After adding GTP, pour the supernatant into ultracentrifugation tubes, and incubate for 45 minutes at 37 degrees Celsius. Once the polymerized supernatant has been centrifuged, check the volume of the supernatant, S4.Then, divide the prepared pipes buffer evenly into tubes with pellets, and resuspend the pellets with a spoon.
After homogenizing the pellets, centrifuge the suspension at 75, 000G for 60 minutes at four degrees Celsius. Measure the volume of the supernatant, S5, and add the calculated volume of dimethyl sulfoxide. Pour the suspension into ultracentrifugation tubes, and incubate for 20 minutes at 37 degrees Celsius to allow polymerization.
Then, centrifuge the polymerized tubulin at 75, 000G for 60 minutes at 30 degrees Celsius. Measure the volume of the supernatant, S6, and keep the pellets aside. Dissolve the pellets in PEM buffer.
Homogenize the pellets using a Dounce glass homogenizer, while adding PEM buffer until completely dissolved.
