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.

<ol>
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The authors have nothing to disclose.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<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>
		</tr>
		<tr>
			<td>1.5- and 2-ml tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>14-ml round-bottom tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15-cm-diameter sterile culture dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15-ml screw-cap tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<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 ...

purification-tubulin-with-controlled-posttranslational-modifications

Research

JoVE Journal

Biochemistry

5.0K Views.  Institut Curie. 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.

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

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

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

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

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

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

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural &#8220;fingerprint&#8221; that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; &#8710;H) of proteins. Comparisons are made between formulations as well as production lots, and differences in derived values indicate differences in thermal stability and structural conformation. Data illustrating the use of DSC in an industrial setting for stability studies as well as monitoring key manufacturing steps are provided as proof of the effectiveness of this protocol. In comparison to other methods for assessing the thermal stability of protein conformations, DSC is cost-effective, requires few sample preparation steps, and also provides a complete thermodynamic profile of the protein unfolding process.

Differential scanning calorimetry measures the thermal transition temperature(s) and total heat energy required to denature a protein. Results obtained are used to assess the thermal stability of protein antigens in vaccine formulations.

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

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

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

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 culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

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

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

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

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

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

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Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

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

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

Ganglioside Extraction, Purification and Profiling