separation and polymerization of tubulin from porcine brain tissue

Efficient Tubulin Isolation from Neural Tissue

Microtubules, hollow protein filaments (24 nm in diameter) formed by alpha- and beta-tubulin heterodimers, are involved in various essential cellular processes. They participate in forming intracellular structures, motility, cell division, cell differentiation, cellular transport, shape maintenance, and secretion1. The cellular functions of microtubules can be affected by direct or indirect interactions with microtubule-associated proteins (MAPs) and other proteins or via complex post-translational modifications defined in the tubulin code2.
Tubulin fibers arise from dynamic non-covalent interactions among alpha- and beta-subunits in the nucleation-elongation mechanism. Short microtubules are formed, and the subsequent growth of tubulin fibers is achieved by reversible elongation at both ends, forming cylinders consisting of tubulin heterodimers arranged in parallel protofilaments2. Dynamic instability refers to the fact that assembled microtubules are often not in equilibrium with their subunits but can undergo phase transition between an extended period of growth and shrinkage while maintaining a steady state1,2.
The dynamic instability of tubulin fibers is principally utilized in many tubulin separation and purification procedures using cycles of high-temperature polymerization and low-temperature depolymerization in the environment of high-purity glycerol, DMSO, GTP/ATP, Mg2+, or other chemical agents (such as taxol or polycations)3. Most of the separation procedures4 are followed by protein chromatography5,6,7,8,9, which ensures the separation of tubulin-associated proteins with nucleoside diphosphokinase and ATPase activity5. Similar results can be obtained by utilizing high-salt buffers10. Multiple sources, including neural11,12,13,14 and non-neural15 tissues, fishes16 (both fresh-water and marine), yeasts, or recombinant variants17 overexpressed in different production strains11,12,13,14, and other sources9,18 were used for fractionation and purification.
The presented protocol utilizes precipitation and protein chromatography to isolate tubulin from the porcine brain into high homogeneity (Figure 1). The main advantage is a relatively high yield achieved with instrumentation available in laboratories equipped for routine molecular biology experiments.

Author Spotlight: Purifying High-Quality Tubulin to Study Protein Dynamics and Therapeutic Applications

Optimizing Tubulin Yield from Porcine Brain Tissue

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 aids in developing targeted brain therapies or drugs.The study of tubulin interactions utilizes advanced methods such as X-ray crystallography, cryo-EM, 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 neural 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 post-translational modification forming the tubulin code that regulates their function, controls cellular function and homeostasis.

Separation and Polymerization of Tubulin from Porcine Brain Tissue

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.

separation-and-polymerization-of-tubulin-from-porcine-brain-tissue

Research

JoVE Journal

Biochemistry

Neural tissue from any source is an excellent material for tubulin isolation, as the dendrites and axons of neurons are rich in microtubules. Here, we present a procedure for extracting tubulin that can be employed, with minor modifications, for neural tissue from multiple sources. In the presented protocol, a new clarification step of the crude lysate has been introduced, which led to a significant reduction in the initial amount of insoluble debris before the first polymerization step occurred. This additional step allowed the processing of additional tissue while using the same instrumentation and, thus, increasing the relative volume of the processed homogenate. The newly introduced step has no significant effect on the quality of purified tubulin, as confirmed by in vitro activity assays and transmission electron microscopy. The described procedure contains all crucial steps, including tissue collection, transport, tissue homogenization, tubulin isolation cycles, and final polishing by ion exchange chromatography using FPLC and subsequent activity measurement assays. The homogeneity of purified tubulin was more than 97%, as confirmed by MS/MS analysis utilizing electrospray ionization and MALDI-TOF.

This protocol describes a technique for the high-yield isolation of tubulin from the porcine brain optimized for small-scale instrumentation. The isolation procedures are complemented by procedures for determining tubulin polymerization activity in vitro using co-sedimentation assays and transmission electron microscopy.

Neural tissue from any source is an excellent material for tubulin isolation, as the dendrites and axons of neurons are rich in microtubules. Here, we ...

Porcine Brain Tissue Homogenization for Tubulin Extraction and Purification

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, 000g for one minute at four degrees Celsius. Retain the supernatant and discard the pellet.