Our current protocol yields over 99%pure tubulin while preserving its natural dynamics. This is essential for studying protein on its own and its interaction with its binding partners. Research on tubulin in the brain helps understand neuronal structure, connectivity, neuroplasticity, or even neurodegenerative diseases, and it adds in developing targeted brain therapies or drugs.
The study of tubulin interactions utilizes advanced methods such as X-ray crystallography, TOEM, and NMR spectroscopy. Among these, NMR uniquely captures tubulin's natural behavior in solution, and this is why it requires protein to remain in the original active state. Precise knowledge of tubulin dynamics can drive future developments by enabling more effective drug design for tubulin-related diseases.
For example, it will enhance the understanding of cell division and aiding in the novel therapies for cancer and neurodegenerative conditions. This insight allows for precise modulation of tubulin function, leading to improved therapeutic outcomes. Microtubules are crucial components of eukaryotic cytoskeleton, involved in various cellular functions.
Despite their similar structure, tubulin proteins undergo posttranslational modification, forming the tubulin code that regulates their function, controls cellular function, and homeostasis. To begin, pour 100 milliliters of the extraction buffer into a one-liter plastic beaker. Determine the weight of the buffer and vessel W1.Take the porcine brain out of the transport vessel.
Using fingers and moderate force, remove the cerebellum, fat, big chunks of white matter, meninges and blood vessels from the brain. Place the stripped brain into the beaker containing 100 milliliters of cold extraction buffer and determine its weight, W2.Add the calculated amount of extraction buffer to the beaker. Transfer the brain tissue with extraction buffer into the pre-chilled kitchen blender and process in four to eight short three-second pulses.
Then, using a high-speed dispersion homogenizer, process the partially homogenized tissue for one minute. After that, leave the suspension on ice for three to five minutes while occasionally mixing with a spoon. Pour the cell suspension into pre-chilled ultracentrifugation tubes and centrifuge the tubes at 50, 000 G for one minute at four degrees Celsius.
Retain the supernatant and discard the pellet. To begin, take the clarified extract of the homogenized porcine brain tissue. Pour the extract into pre-chilled ultracentrifugation tubes and centrifuge at 75, 000 G for 60 minutes at four degrees Celsius.
Collect the supernatant and measure its volume, S1.Next, add 10X 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, 000 G 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 Dounce glass homogenizer and homogenize the solution intermittently every 10 minutes for 45 minutes on ice.
Pour the homogenized solution into pre-chilled ultracentrifugation tubes. Centrifuge at 75, 000 G 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, 000 G 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, 000 G 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.
