Name: purification of tubulin with controlled posttranslational modifications and isotypes from limited sources by polymerization-depolymerization cycles
Uploaded: 2020-11-05T15:00:00
Description: Watch this Scientific Journal Video about Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles at JoVE.co

This work was supported by the ANR-10-IDEX-0001-02, the LabEx Cell&#8217;n&#8217;Scale ANR-11-LBX-0038 and the Institut de convergence Q-life ANR-17-CONV-0005. CJ is supported by the Institut Curie, the French National Research Agency (ANR) awards ANR-12-BSV2-0007 and ANR-17-CE13-0021, the Institut National du Cancer (INCA) grant 2014-PL BIO-11-ICR-1, and the Fondation pour la Recherche Medicale (FRM) grant DEQ20170336756. MMM is supported by the Fondation Vaincre Alzheimer grant FR-16055p, and by the France Alzheimer grant AAP SM 2019 n&#176;2023. JAS was supported by the European Union&#8217;s Horizon 2020 research and innovation program under the Marie Sk&#322;odowska-Curie grant agreement No 675737, and the FRM grant FDT201904008210. SB was supported by the FRM grant FDT201805005465.
We thank all members of the Janke lab, in particular J. Souphron, as well as G. Lakisic (Institut MICALIS, AgroParisTech) and A. Gautreau (Ecole Polytechnique) for help during the establishment of the protocol. We would like to thank the animal facility of the Institut Curie for help with mouse breeding and care.
The antibody 12G10, developed by J. Frankel and M. Nelson, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa.

Animal care and use for this study were performed in accordance with the recommendations of the European Community (2010/63/UE). Experimental procedures were specifically approved by the ethics committee of the Institut Curie CEEA-IC #118 (authorization no. 04395.03 given by National Authority) in compliance with the international guidelines.
1. Preparation of Reagents for Tubulin Purification
NOTE: All the buffers used for tubulin purification should contain potassiu...

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The method described here provides a platform to rapidly generate high-quality, assembly-competent tubulin in medium-large quantities from cell lines and single mouse brains. It is based on the gold-standard protocol of tubulin purification from bovine brains used in the field for many years16,17. One particular advantage of the approach is the use of suspension cultures of HeLa S3 cells, which, once established, yields large amounts of cells while requiring litt...

Microtubules play critical roles in many cellular processes. They give cells their shape, build meiotic and mitotic spindles for chromosome segregation, and serve as tracks for intracellular transport. To perform these diverse functions, microtubules organize themselves in different ways. One of the intriguing questions in the field is to understand the molecular mechanisms that allow the structurally and evolutionarily conserved microtubules to adapt to this plethora of organizations and functions. One potential mechanism is the diversification of microtubules, which is defined by the concept known as the &#8216;tubulin code&#8217;1,2,3. The tubulin code includes two principal components: differential incorporation of &#945;- and &#946;-tubulin gene products (tubulin isotypes) into the microtubules and tubulin posttranslational modifications (PTMs).
Since the 1970s, in vitro reconstitution experiments, combined with evolving light microscopy techniques, have paved the way for important discoveries about the properties of microtubules: dynamic instability4 and treadmilling5, and their other mechanisms and functions6,7,8,9,10,11,12,13,14,15. Almost all the in vitro experiments performed so far have been based on tubulin purified from brain tissue using repeated cycles of polymerization and depolymerization16,17. Although purification from the brain tissue confers the advantage of obtaining high-quality tubulin in large quantities (usually gram amounts), one important drawback is the heterogeneity as tubulin purified from brain tissue is a mixture of different tubulin isotypes and is enriched with many tubulin PTMs. This heterogeneity makes it impossible to delineate the role of a particular tubulin PTM or isotype in the control of microtubule properties and functions. Thus, producing assembly-competent tubulin with controlled tubulin PTMs and homogenous isotype composition is essential to address the molecular mechanisms of the tubulin code.
Recently, an approach to purify tubulin by affinity chromatography using the microtubule-binding TOG (tumor-overexpressed gene) domain of yeast Stu2p has been developed18. In this method, tubulin in crude lysates of cells or tissue is passed through a column where it binds to the matrix-immobilized TOG domain, which allows the analysis of the whole tubulin pool of a given, even very small, sample. A long-awaited approach to purify recombinant tubulin has also been described in recent years. It is based on the baculovirus system, in which a bi-cistronic vector containing &#945;- and&#160;&#946;-tubulin genes is expressed in insect cells19. However, this method is very cumbersome and time-consuming and is therefore mostly used for studying the impact of tubulin mutations20 and tubulin isotypes21,22,23 in vitro.
In the current protocol, we describe a method that uses the well-established and widely used polymerization-depolymerization approach as a blueprint to generate tubulin with different levels of modification either from cell lines or from mouse brain tissue24. In this procedure, tubulin is cycled between the soluble (tubulin dimer at 4 &#176;C) and polymerized form (microtubule at 30 &#176;C in the presence of guanosine 5&#39;-triphosphate [GTP]). Each form is separated through successive steps of centrifugation: tubulin dimers will remain in the supernatant after a cold (4 &#176;C) spin, whereas microtubules will be pelleted at 30 &#176;C. Furthermore, one polymerization step is carried out at high piperazine-N,N&#8242;-bis(2-ethanesulfonic acid) (PIPES) concentration, which allows the removal of microtubule-associated proteins from the microtubules and thus, from the finally purified tubulin. Tubulin purified from HeLa S3 cells grown as suspension or adherent cultures is virtually free of any tubulin PTM and has been used in recent in vitro reconstitution experiments25,26,27,28. We have further adapted the method to purify tubulin from single mouse brains, which can be used for a large number of mouse models with changes in tubulin isotypes and PTMs.
In the protocol, we first describe the generation of the source material (cell mass or brain tissue), its lysis (Figure 1A), followed by the successive steps of tubulin polymerization and depolymerization to purify the tubulin (Figure 1B). We further describe the process to assess the purity (Figure 2A,B) and quantity (Figure 3A,B) of the purified tubulin. The method can be adapted to produce tubulin enriched with a selected PTM by overexpressing a modifying enzyme in cells prior to tubulin purification (Figure 4B). Alternatively, tubulin-modifying enzymes can be added to tubulin during the purification process. Finally, we can purify tubulin lacking specific isotypes or PTMs from the brains of mice deficient in the corresponding tubulin-modifying enzymes (Figure 4B)29.
The method we describe here has two main advantages: (i) it allows the production of sufficiently large amounts of tubulin in a relatively short time, and (ii) it generates high-quality, pure tubulin, with either specific tubulin isotype composition or PTMs. In the associated video of this manuscript, we highlight some of the critical steps involved in this procedure.

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			<td>1&#160;M MgCl2&#160;</td>
			<td>Sigma</td>
			<td>#M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>1-L cell culture vessels</td>
			<td>Techne F7610&#160;</td>
			<td></td>
			<td>Used for spinner cultures. Never stir the empty spinner bottles. When spinner bottles are in the cell culture incubator, always keep the lateral valves of spinner bottles slightly open to facilitate the equilibration of media with incubator&#8217;s atmosphere. After use, fill the spinner bottles immediately with tap water to avoid drying of remaining cells on the bottle walls. Wash the bottles with deionised water, add app 200 ml of deionised water and autoclave. Under a sterile cell culture hood remove the water and allow the bottles to dry completely, still under the hood, for several hours. Never use detergents for cleaning the spinner bottles because any trace amounts of the detergent can be deleterious to the cells.</td>
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			<td>1.5- and 2-ml tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>14-ml round-bottom tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>15-cm-diameter sterile culture dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>15-ml screw-cap tubes</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>2-mercaptoethanol&#160;</td>
			<td>Sigma&#160;</td>
			<td>#M3148</td>
			<td>2-mercaptoethanol is toxic and should be used under the hood.</td>
		</tr>
		<tr>
			<td>4-(2-aminoethyl)-benzenesulfonyl fluoride&#160;</td>
			<td>Sigma&#160;</td>
			<td>#A8456</td>
			<td></td>
		</tr>
		<tr>
			<td>40% Acrylamide&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>#161-0140</td>
			<td></td>
		</tr>
		<tr>
			<td>5-, 10- 20-ml syringes</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>5-ml, 10-ml, 25-ml sterile pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50-ml screw-cap tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma</td>
			<td>#A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-alpha-tubulin antibody, 12G10&#160;</td>
			<td>Developed by J. Frankel and M. Nelson, obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD, and maintained by the University of Iowa</td>
			<td></td>
			<td>dilution: 1/500</td>
		</tr>
		<tr>
			<td>Anti-glutamylated tubulin antibody, GT335&#160;</td>
			<td>AdipoGen&#160;</td>
			<td>#AG-20B-0020</td>
			<td>dilution: 1/20,000</td>
		</tr>
		<tr>
			<td>Aprotinin&#160;</td>
			<td>Sigma&#160;</td>
			<td>#A1153</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance (0.1 &#8211; 10 g)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman 1-l polypropylene bottles&#160;</td>
			<td></td>
			<td></td>
			<td>For collecting spinner cultures</td>
		</tr>
		<tr>
			<td>Beckman Avanti J-26 XP centrifuge</td>
			<td></td>
			<td></td>
			<td>For collecting spinner cultures</td>
		</tr>
		<tr>
			<td>Biological stirrer&#160;</td>
			<td>Techne MCS-104L&#160;</td>
			<td></td>
			<td>Installed in the cell culture incubator (for spinner cultures), 25 rpm for Hela S3 and HEK 293 cells</td>
		</tr>
		<tr>
			<td>Bis N,N&#8217;-Methylene-Bis-Acrylamide&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>#161-0201</td>
			<td></td>
		</tr>
		<tr>
			<td>Blender IKA Ultra-Turrax&#174;&#160;</td>
			<td></td>
			<td></td>
			<td>For lysing brain tissue, use 5-mm probe, with the machine set at power 6 or 7. Blend the brain tissue 2-3 times for 15 s on ice.</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma&#160;</td>
			<td>#A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue&#160;</td>
			<td>Sigma&#160;</td>
			<td>#1.08122</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxypeptidase A (CPA)</td>
			<td>Sigma</td>
			<td>#C9268</td>
			<td>Concentration: 1.7&#160;U/&#181;l</td>
		</tr>
		<tr>
			<td>Cell culture hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator set at 37&#176;C, 5% CO2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>&#160;Sigma&#160;</td>
			<td>#D8418</td>
			<td>DMSO can enhance cell and skin permeability of other compounds. Avoid contact and use skin and eye protection.</td>
		</tr>
		<tr>
			<td>DMEM medium&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#41965062</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT, DL-Dithiothreitol&#160;</td>
			<td>Sigma&#160;</td>
			<td>#D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Euromedex</td>
			<td>#EU0007-C</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma&#160;</td>
			<td>#E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute&#160;</td>
			<td>Fisher Chemical&#160;</td>
			<td>#E/0650DF/15</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma&#160;</td>
			<td>#F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>French pressure cell press&#160;</td>
			<td>Thermo electron corporation</td>
			<td>&#160;#FA-078A</td>
			<td>with a #FA-032 cell; for lysing big amounts of cells. Set at medium ratio, and the gauge pressure of 1,000 psi (corresponds to 3,000 psi inside the disruption chamber).</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>&#160;VWR Chemicals&#160;</td>
			<td>#24388.295</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>#G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP&#160;</td>
			<td>Sigma&#160;</td>
			<td>#G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating block&#160;</td>
			<td>Stuart&#160;</td>
			<td>#SBH130D</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela cells&#160;</td>
			<td></td>
			<td>ATCC&#174; CCL-2&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela S3 cells&#160;</td>
			<td>ATCC</td>
			<td>ATCC&#174; CCL-2.2&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl )</td>
			<td>VWR</td>
			<td>#20252.290</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope&#160;</td>
			<td></td>
			<td></td>
			<td>With fluorescence if cell transfection is to be verified</td>
		</tr>
		<tr>
			<td>Isopropanol&#160;</td>
			<td>VWR&#160;</td>
			<td>#20842.298</td>
			<td></td>
		</tr>
		<tr>
			<td>jetPEI</td>
			<td>Polyplus&#160;</td>
			<td>#101</td>
			<td></td>
		</tr>
		<tr>
			<td>JLA-8.1000 rotor&#160;</td>
			<td></td>
			<td></td>
			<td>For collecting spinner cultures</td>
		</tr>
		<tr>
			<td>KOH&#160;</td>
			<td>Sigma&#160;</td>
			<td>#P1767</td>
			<td>KOH is corrosive and causes burns; use eye and skin protection.</td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#25030123</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory centrifuge for 50-ml tubes</td>
			<td>Sigma</td>
			<td>4-16 K&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin&#160;</td>
			<td>Sigma</td>
			<td>#L2884</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-pipettes p2.5, p10, p20, p100, p200 and p1000 and corresponding tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropestles</td>
			<td>Eppendorf</td>
			<td>#0030 120.973</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse brain tissue&#160;</td>
			<td></td>
			<td></td>
			<td>Animal care and use for this study were performed in accordance with the recommendations of the European Community (2010/63/UE). Experimental procedures were specifically approved by the ethics committee of the Institut Curie CEEA-IC #118 (authorization no. 04395.03 given by National Authority) in compliance with the international guidelines.</td>
		</tr>
		<tr>
			<td>Needles 18G X 1 &#189;&#8221; (1.2 X 38 mm</td>
			<td>Terumo</td>
			<td>#18G</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles 20G X 1 &#189;&#8221; (0.9 X 38 mm</td>
			<td>Terumo</td>
			<td>#20G</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles 21G X 4 &#190;&#8221; (0.8 X 120 mm</td>
			<td>B.Braun</td>
			<td>#466 5643</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#15140130</td>
			<td></td>
		</tr>
		<tr>
			<td>pH-meter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Sigma&#160;</td>
			<td>#P7626</td>
			<td>PMSF powder is hazardous. Use skin and eye protection when preparing PMSF solutions.</td>
		</tr>
		<tr>
			<td>PIPES&#160;</td>
			<td>Sigma&#160;</td>
			<td>#P6757</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette-boy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotors</td>
			<td>Beckman 70.1 Ti; TLA-100.3; and TLA 55</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE electrophoresis equipment&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>#1658001FC</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS, Sodium dodecyl sulphate&#160;</td>
			<td>VWR&#160;</td>
			<td>#442444H</td>
			<td>For preparing Laemmeli buffer&#160;</td>
		</tr>
		<tr>
			<td>SDS, Sodium dodecyl sulphate&#160;</td>
			<td>Sigma&#160;</td>
			<td>#L5750</td>
			<td>For preparing &#39;TUB&#39; SDS-PAGE gels</td>
		</tr>
		<tr>
			<td>Sonicator&#160;</td>
			<td>Branson</td>
			<td>#101-148-070</td>
			<td>Used for lysing cells grown as adherent cultures. Use 6.5 mm diameter probe, set the sonicator at &#8220;Output control&#8221; 1, &#8220;Duty cycle&#8221; 10% and time depending on the cell type used.</td>
		</tr>
		<tr>
			<td>Tabletop centrifuge for 1.5 ml tubes</td>
			<td>Eppendorf</td>
			<td>5417R&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED, N, N, N&#8242;, N&#8242;-Tetramethylethylenediamine&#160;</td>
			<td>Sigma</td>
			<td>#9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichostatin A (TSA)</td>
			<td>Sigma</td>
			<td>#T8552</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma&#160;</td>
			<td>#T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base (Tris)</td>
			<td>Sigma&#160;</td>
			<td>#T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Life Technologies</td>
			<td>#15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotors&#160;</td>
			<td></td>
			<td>TLA-55, TLA-100.3 and 70.1 Ti rotors</td>
			<td>Set at 4&#176;C or 30&#176;C based on the need of the experiment&#160;</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman</td>
			<td>#357448&#160;</td>
			<td>for using with TLA-55 rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman</td>
			<td>#349622</td>
			<td>for using with TLA-100.3 rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman</td>
			<td>#355631&#160;</td>
			<td>for using with 70.1 Ti rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuges</td>
			<td>Beckman</td>
			<td>Optima L80-XP (or equivalent) and Optima MAX-XP (or equivalent)</td>
			<td>Set at 4&#176;C or 30&#176;C based on the need of the experiment&#160;</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath equipped with floaters or tube holders</td>
			<td></td>
			<td></td>
			<td>Set at 30&#176;C&#160;</td>
		</tr>
	</tbody>
</table>

purification of tubulin with controlled posttranslational modifications and isotypes from limited sources by polymerization-depolymerization cycles

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They allow the analysis of the intrinsic properties of microtubules, such as dynamics, and their interactions with microtubule-associated proteins (MAPs). The &#8220;tubulin code&#8221; is an emerging concept that points to different tubulin isotypes and various posttranslational modifications (PTMs) as regulators of microtubule properties and functions. To explore the molecular mechanisms of the tubulin code, it is crucial to perform in vitro reconstitution experiments using purified tubulin with specific isotypes and PTMs.
To date, this was technically challenging as brain tubulin, which is widely used in in vitro experiments, harbors many PTMs and has a defined isotype composition. Hence, we developed this protocol to purify tubulin from different sources and with different isotype compositions and controlled PTMs, using the classical approach of polymerization and depolymerization cycles. Compared to existing methods based on affinity purification, this approach yields pure, polymerization-competent tubulin, as tubulin resistant to polymerization or depolymerization is discarded during the successive purification steps.
We describe the purification of tubulin from cell lines, grown either in suspension or as adherent cultures, and from single mouse brains. The method first describes the generation of cell mass in both suspension and adherent settings, the lysis step, followed by the successive stages of tubulin purification by polymerization-depolymerization cycles. Our method yields tubulin that can be used in experiments addressing the impact of the tubulin code on the intrinsic properties of microtubules and microtubule interactions with associated proteins.

The main goal of this method is to produce high-quality, assembly-competent tubulin in quantities sufficient to perform repeated in vitro experiments with the purified components. Microtubules assembled from this tubulin can be used in reconstitution assays based on the total internal reflection fluorescence (TIRF) microscopy technique with either dynamic or stable microtubules, in experiments testing microtubule dynamics, interactions with MAPs or molecular motors, and force generation by the motors25<...

Watch this Scientific Journal Video about Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles at JoVE.co

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

This protocol describes tubulin purification from small/medium-scale sources such as cultured cells or single mouse brains, using polymerization and depolymerization cycles. The purified tubulin is enriched in specific isotypes or has specific posttranslational modifications and can be used in in vitro reconstitution assays to study microtubule dynamics and interactions.

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They ...

This method allows to generate pure assembly-competent tubulin with defined post-translational modifications in quantities sufficient to perform entire sets of in-vitro experiments. This protocol based on cycles of tubulin polymerization and de-polymerization is easy to perform in any cell biology lab. It allows to generate good amounts of tubulin with very little hands-on time.To purify tubulin from suspension cultures, nine days before the tubulin prep, revive and amplify the desired cell type to obtain six confluent 15 centimeter dishes. Eight days before the tubulin prep, add one liter of pre-warmed medium to every spinner bottle per dish under a cell culture cabinet and place the spinners on a stirring table inside the cell culture incubator. Leave the lateral spinner caps slightly open to allow the medium to equilibrate to the atmosphere of the incubator.The next day, use the cells from the confluent dishes to inoculate the spinner bottles. Return the spinner bottles to the incubator for one week with the lateral valves slightly open. To harvest the cells, transfer the cultures from the spinner bottles into one liter centrifuge bottles and collect cells by centrifugation.Resuspend each pellet in 10 milliliters of ice-cold PBS, and pool the resuspended cells in a 50 milliliter tube for centrifugation at four degrees Celsius. For a 10 milliliters cell pellet, add 10 milliliters of lysis buffer to the pellet, and invert the tube several times to re-suspend the cells. To purify tubulin from adherent cell cultures, revive and amplify the desired cell type to obtain 10 confluent 15 centimeter dishes on the day of the tubulin prep.To harvest the adherent cells, treating three dishes at a time, tilt the culture dishes to remove the medium while a second person gently washes the cells with seven milliliters of PBS-EDTA. After the washing, the second person should treat the cells with five milliliters of PBS-EDTA per dish before having a third person use a cell lifter to gently push the cells to one edge of the dish. When the cells have been detached, pool the cells from all three dishes into a 50 milliliter tube on ice and rinse each plate with an additional two milliliters of PBS-EDTA to collect any remaining cells.After collecting the cells by centrifugation, discard the supernatant to determine the volume of the cell pellet. Add the lysis buffer, re-suspend the cells, and transfer them into a 14 milliliter round bottom tube. Sonicate the cells for approximately 45 pulses.For mouse brain lysis, add 500 microliters of licensed buffer to a single brain harvested from an adult mouse and use a tissue blender to lyse the tissue. After obtaining the lysate at a 1/100th of its volume to an equal volume of 2X Laemmli buffer and boil the solution for five minutes. Store it at minus 20 degrees Celsius until quantification.Collect a sample this way at each step of the tubulin prep. To clarify the lysate, centrifuge the sample and use a long needle to carefully transfer the clear supernatant to a new tube, taking care to avoid the upper floating layer. After the lysate clarification, it is essential to remove the floating material in the supernatant, otherwise it could negatively affect the efficiency of tubulin purification.To polymerize tubulin from the clarified lysate, mix the entire volume of supernatant one with a 1/200th volume of 0.2 molar GTP and half a volume of warm glycerol in an appropriately sized tube. Gently pipette the mixture, take care to avoid introducing air bubbles. Seal the tubes with parafilm and place them in a 30 degrees Celsius water bath for 20 minutes.After centrifugation, transfer the supernatant to a new tube. To de-polymerize the microtubules present in the pellet two, resuspend the pellet in ice cold BRB 80 for five minutes on ice. Pipette the sample first with a P1000 pipette, then with a P200 pipette, every five minutes for 20 minutes on ice.Once the sample is homogenous, centrifuge it and transfer the obtained supernatant to a new tube. To remove microtubule associated proteins from the tubulin, mix the entire volume of supernatant three with an equal volume of preheated one molar pipe S, the same volume of preheated glycerol and a 1/100th volume of 0.2 molar GTP. Incubate the mix for 20 minutes at 30 degrees Celsius before centrifuging.Transfer the microtubule-associated protein-containing supernatant four into a new tube, the pellet contains polymerized microtubules. After performing a third polymerization and de-polymerization, the amount of harvested tubulin can be quantified by SDS page analysis. The success of the purification process can be confirmed on a Coomassie stained SDS page.A lower than expected yield of the purified tubulin can be due to an inefficient tubulin polymerization as evidenced by lower amount of tubulin in the second, fourth and sixth pellet fractions and higher amount in the second, fourth and sixth supernatant fractions. To quantify the purified tubulin, the samples can be run next to known quantities of bovine serum albumin. Quantitative densitometry of the protein bands then allows to calculate the quantity of purified tubulin.The modification status of the tubulin can be confirmed by immuno blot analysis of the samples. Our protocol allows the isolation of tubulin with controlled post-translational modifications. With this tubulin, we can set up in vitro reconstitution experiments that allow us to determine the impact of tubulin modifications on micro-tubal properties, as well as on their interactions with microtubule associated proteins.

Research

JoVE Journal

Biochemistry

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Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

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A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

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Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

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Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

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Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

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Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

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Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

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