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 allow the analysis of the intrinsic properties of microtubules, such as dynamics, and their interactions with microtubule-associated proteins (MAPs). The “tubulin code” 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.
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 ‘tubulin code’1,2,3. The tubulin code includes two principal components: differential incorporation of α- and β-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 α- and β-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 °C) and polymerized form (microtubule at 30 °C in the presence of guanosine 5'-triphosphate [GTP]). Each form is separated through successive steps of centrifugation: tubulin dimers will remain in the supernatant after a cold (4 °C) spin, whereas microtubules will be pelleted at 30 °C. Furthermore, one polymerization step is carried out at high piperazine-N,N′-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.
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 potassium salts and NOT sodium salts30.
2. Amplification and harvesting sources of tubulin
NOTE: In this protocol, three sources of tubulin were used: (i) cells (HeLa S3 and HEK-293) grown as suspension cultures; (ii) cells grown as adherent cultures (HEK-293, HeLa, and U2 OS); and (iii) mouse brain tissue. This protocol considers the day of tubulin purification as ‘day 0’ and accordingly, other steps have been described relative to day 0.
3. Lysis of Cells or Brain Tissue
4. Purification of Tubulin
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. They can also be used in microtubule-MAP co-pelleting assays and solid-state NMR spectroscopy28.
The enrichment and purity of tubulin throughout the purification process can be monitored by using a Coomassie-stained SDS-polyacrylamide gel electrophoresis (PAGE) gel, preferably the ‘TUB’ SDS-PAGE gels, that allow for the separation of α- and β-tubulins, which co-migrate as a single band in classical gels32. Lysates collected at different steps (except for the very last depolymerization, see protocol) are loaded onto the gel in comparable amounts for assessing the success of tubulin purification (Figure 2A)24. The final tubulin sample, which is very precious, is only loaded on the gel for the determination of tubulin concentration. It is normal to lose some tubulin in the process of repeated cycles of polymerization and depolymerization. A lower-than-expected yield of the final purified tubulin can be due to either (i) incomplete depolymerization of microtubules, visualized by the presence of an important amount of tubulin in fractions P3, P5, and P7, or (ii) an inefficient tubulin polymerization into microtubules, in which case a lower amount of tubulin is present in fractions P2, P4, and P6 and higher in fractions SN2, SN4, and SN6 (Figure 2B). If the tubulin is lost during polymerization steps (lower amounts of P2 and P4) (i) ensure sufficient tubulin concentration during polymerization (ii) use a fresh aliquot of GTP, and/or (iii) reconfirm the temperature of the polymerization reaction. If the tubulin is lost during depolymerization steps (lower amounts of SN3 and SN5), increase the time as well as pipetting of the mix on ice.
For the quantification of purified tubulin, run the samples along with the known quantities of bovine serum albumin (BSA, 0.5 µg – 1 µg – 2 µg – 4 µg) (Figure 3A) on SDS-PAGE. Gels are stained with Coomassie brilliant blue, scanned, and the intensities of BSA and tubulin bands are measured by quantitative densitometry (Figure 3B) as described at https://openwetware.org/wiki/Protein_Quantification_Using_ImageJ. Please note that the same analysis can be done in Fiji, an upgraded version of ImageJ33. Values from the BSA bands were used to determine the linear regression equation, which was used to calculate the amount of protein in the tubulin bands. Only tubulin band intensities within the range of the BSA curve are used to determine tubulin concentration. Based on the calculated tubulin concentration, aliquots of desired volumes of tubulin are prepared, snap-frozen in liquid nitrogen, and stored at -80 °C. We usually obtain about ~2 mg of tubulin from four spinner bottles of HeLa S3 suspension cultures (~15 g of cells), ~250 µg of tubulin from ten 15-cm diameter dishes (~1.2 g of cells), and ~1 mg of tubulin from 1 g of mouse brain tissue.
To confirm the enrichment of a particular tubulin isotype or modification, ~0.1 µg of the purified tubulin can be immunoblotted using respective antibodies34,35. The control tubulin will vary depending on the tubulin of interest. For tubulin modified in vitro with a modifying enzyme, use non-treated tubulin as control. For tubulin modified in cellulo by the overexpression of a modifying enzyme, use tubulin purified from cells that do not express the enzyme as control (Figure 4A). Control tubulin for tubulin purified from knockout-mouse brains will be tubulin from wild type mice (Figure 4B). In all immunoblot analyses, an equal load of tubulin is verified by using a PTM-independent anti-α-tubulin antibody (12G10).
Figure 1: Tubulin purification from different sources using polymerization-depolymerization cycles. (A) Different sources of tubulin are lysed using specific strategies. HeLa S3 cells cultured in suspension are lysed using a French press; HEK-293 cells are lysed by repetitive pipetting. Adherent cells were lysed using short pulses of sonication and mouse brain tissue using a tissue homogenizer. (B) Schematic representation of the successive steps of the tubulin purification protocol using cycles of cold-depolymerization and warm-polymerization. After lysis and lysate clarification, microtubules are polymerized and pelleted. Microtubules are then depolymerized and subsequently allowed to polymerize in a high-molarity buffer, preventing microtubule-associated protein (MAP) co-sedimentation with the microtubules. MAP-free microtubules are then depolymerized and can be further subjected to a third cycle of polymerization-depolymerization to remove trace amounts of the high-molarity buffer. Please click here to view a larger version of this figure.
Figure 2: Evaluating the success of the tubulin purification. Samples collected at different steps of the tubulin purification protocol were run on a ‘TUB’ sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (see protocol for details) and stained with Coomassie brilliant blue. (A) In a successful tubulin purification, α- and β-tubulins are progressively enriched throughout the process. After the second polymerization, the microtubule pellet (P4) is virtually free of contamination from other proteins or microtubule-associated proteins (MAPs). Note that it is normal to lose some tubulin during the procedure. (B) In an unsuccessful tubulin purification, the final tubulin yield is low, and tubulin remains either in the pellet after depolymerization or in the supernatant after polymerization (red boxes). In the example shown here, tubulin did not polymerize efficiently in both polymerization steps. Please click here to view a larger version of this figure.
Figure 3: Quantification of the purified tubulin using Coomassie-stained sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and densitometry. (A) Coomassie-stained SDS-PAGE gel with known quantities of bovine serum albumin (BSA; 0.5, 1, 2 and 4 µg, gray gradient line) and different volumes (0.5 and 1 µL, light and dark colors, respectively) of purified tubulin. In the example shown, tyrosinated tubulin (HeLa S3 tubulin, light and dark orange) and detyrosinated tubulin (HeLa S3 tubulin treated with carboxypeptidase A, light and dark blue) were loaded on the gel. (B) BSA bands from (A) were quantified using ImageJ (in arbitrary units, AU) and plotted against the amount of protein loaded (gray to black points). Those points were used to calculate the linear regression line (the gray gradient line) and equation, which were used to calculate the amounts of protein in the tubulin samples (light and dark orange and blue points) loaded on the gel. This facilitated the calculation of the concentration of the tubulin samples. Note that the points that lie beyond the BSA standard curve should not be used to determine concentration (dark orange and blue points). Please click here to view a larger version of this figure.
Figure 4: Immunoblot analysis of purified tubulin with different PTMs. (A) Tubulins purified from HEK-293 cells: wild type, or cells overexpressing TTLL5 or TTLL7 were analyzed for the specific enrichment of polyglutamylation using the GT335 antibody. While TTLL5 overexpression increases polyglutamylation on α- and β-tubulin, TTLL7 overexpression specifically enriches β-tubulin glutamylation. (B) Tubulin purified from brain tissues of wild type and ttll1-/- mice were analyzed for patterns of glutamylation. Note the strong reduction of polyglutamylation of tubulin from ttll1-/- mice, which lack the major brain glutamylase TTLL136. ‘TUB’ gels were used to separate α- and β-tubulin. An equal amount of tubulin load was confirmed by 12G10, an anti-α-tubulin antibody. Please click here to view a larger version of this figure.
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 little hands-on time. This makes the protocol relatively easy to perform in any cell biology lab, whereas other tubulin purification methods18,19,32,37 require specific equipment and expertise and are thus mostly used by laboratories with a strong background in protein purification. When producing smaller quantities of tubulin from adherent cell lines, a variety of cell lines can be used. We have successfully purified tubulin from HeLa, U-2 OS, and HEK-293 cells. If a larger-scale purification is needed, harvested cells or brains can be snap-frozen in lysis buffer and stored at -80 °C, and multiple cell pellets or brains can be pooled together to purify larger amounts of tubulin.
Tubulin purified from cell lines is virtually free of tubulin PTMs. This Tyr-tubulin can readily be converted to detyrosinated (deTyr-) tubulin in a single straightforward step25. To produce tubulin with other PTMs, specific tubulin-modifying enzymes can be overexpressed in cells prior to tubulin purification. Furthermore, using cell lines of human origin as the source of material helps to avoid potential cross-species issues when studying interactions between microtubules and human MAPs. Further, tubulin from untransformed (such as HEK293) or transformed (such as HeLa) cells can provide information about the effects of microtubule-directed drugs (e.g., taxanes) on normal- vs. tumor-cell microtubules.
Finally, our protocol facilitates the purification of tubulin from single mouse brains. As an increasing number of mouse models of tubulin mutations and modifications are being generated, this protocol allows direct analysis of the properties and interactions of microtubules with altered tubulin isotype composition38,39,40 or tubulin PTMs31,41.
The approach is based on cycles of polymerization and depolymerization. Thus, specific tubulin isotypes or tubulin with particular PTMs that affect the assembly and disassembly properties of microtubules could result in a disproportionate loss or reduction of such tubulin forms during the purification process. Nevertheless, we have shown that major tubulin PTMs, such as acetylation, detyrosination, glutamylation, and glycylation, are retained on the microtubules throughout the tubulin purification process24. However, it should be noted that for quantitative analyses of the tubulin composition in cells or tissues, the TOG-column-based tubulin purification approach is more appropriate as it would allow an unbiased, polymerization-independent tubulin purification18. Despite its limitation, our protocol offers a great advantage in generating large amounts of high-quality tubulin that can be used in meticulous in vitro reconstitution experiments. In particular, it facilitates the use of PTM-rich brain tubulin in routine experiments.
The authors have nothing to disclose.
This work was supported by the ANR-10-IDEX-0001-02, the LabEx Cell’n’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°2023. JAS was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-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.
1 M MgCl2 | Sigma | #M1028 | |
1-L cell culture vessels | Techne F7610 | 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’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. | |
1.5- and 2-ml tubes | |||
14-ml round-bottom tubes | |||
15-cm-diameter sterile culture dishes | |||
15-ml screw-cap tubes | |||
2-mercaptoethanol | Sigma | #M3148 | 2-mercaptoethanol is toxic and should be used under the hood. |
4-(2-aminoethyl)-benzenesulfonyl fluoride | Sigma | #A8456 | |
40% Acrylamide | Bio-Rad | #161-0140 | |
5-, 10- 20-ml syringes | |||
5-ml, 10-ml, 25-ml sterile pipettes | |||
50-ml screw-cap tubes | |||
Ammonium persulfate (APS) | Sigma | #A3678 | |
Anti-alpha-tubulin antibody, 12G10 | 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 | dilution: 1/500 | |
Anti-glutamylated tubulin antibody, GT335 | AdipoGen | #AG-20B-0020 | dilution: 1/20,000 |
Aprotinin | Sigma | #A1153 | |
Balance (0.1 – 10 g) | |||
Beckman 1-l polypropylene bottles | For collecting spinner cultures | ||
Beckman Avanti J-26 XP centrifuge | For collecting spinner cultures | ||
Biological stirrer | Techne MCS-104L | Installed in the cell culture incubator (for spinner cultures), 25 rpm for Hela S3 and HEK 293 cells | |
Bis N,N’-Methylene-Bis-Acrylamide | Bio-Rad | #161-0201 | |
Blender IKA Ultra-Turrax® | 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. | ||
Bovine serum albumin (BSA) | Sigma | #A7906 | |
Bromophenol blue | Sigma | #1.08122 | |
Carboxypeptidase A (CPA) | Sigma | #C9268 | Concentration: 1.7 U/µl |
Cell culture hood | |||
Cell culture incubator set at 37°C, 5% CO2 | |||
Dimethyl sulfoxide (DMSO) | Sigma | #D8418 | DMSO can enhance cell and skin permeability of other compounds. Avoid contact and use skin and eye protection. |
DMEM medium | Life Technologies | #41965062 | |
DTT, DL-Dithiothreitol | Sigma | #D9779 | |
EDTA | Euromedex | #EU0007-C | |
EGTA | Sigma | #E3889 | |
Ethanol absolute | Fisher Chemical | #E/0650DF/15 | |
Fetal bovine serum (FBS) | Sigma | #F7524 | |
French pressure cell press | Thermo electron corporation | #FA-078A | 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). |
Glycerol | VWR Chemicals | #24388.295 | |
Glycine | Sigma | #G8898 | |
GTP | Sigma | #G8877 | |
Heating block | Stuart | #SBH130D | |
Hela cells | ATCC® CCL-2™ | ||
Hela S3 cells | ATCC | ATCC® CCL-2.2™ | |
Hydrochloric acid (HCl ) | VWR | #20252.290 | |
Inverted microscope | With fluorescence if cell transfection is to be verified | ||
Isopropanol | VWR | #20842.298 | |
jetPEI | Polyplus | #101 | |
JLA-8.1000 rotor | For collecting spinner cultures | ||
KOH | Sigma | #P1767 | KOH is corrosive and causes burns; use eye and skin protection. |
L-Glutamine | Life Technologies | #25030123 | |
Laboratory centrifuge for 50-ml tubes | Sigma | 4-16 K | |
Leupeptin | Sigma | #L2884 | |
Liquid nitrogen | |||
Micro-pipettes p2.5, p10, p20, p100, p200 and p1000 and corresponding tips | |||
Micropestles | Eppendorf | #0030 120.973 | |
Mouse brain tissue | 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. | ||
Needles 18G X 1 ½” (1.2 X 38 mm | Terumo | #18G | |
Needles 20G X 1 ½” (0.9 X 38 mm | Terumo | #20G | |
Needles 21G X 4 ¾” (0.8 X 120 mm | B.Braun | #466 5643 | |
Parafilm | |||
PBS | Life Technologies | #14190169 | |
Penicillin-Streptomycin | Life Technologies | #15140130 | |
pH-meter | |||
Phenylmethanesulfonyl fluoride (PMSF) | Sigma | #P7626 | PMSF powder is hazardous. Use skin and eye protection when preparing PMSF solutions. |
PIPES | Sigma | #P6757 | |
Pipette-boy | |||
Rotors | Beckman 70.1 Ti; TLA-100.3; and TLA 55 | ||
SDS-PAGE electrophoresis equipment | Bio-Rad | #1658001FC | |
SDS, Sodium dodecyl sulphate | VWR | #442444H | For preparing Laemmeli buffer |
SDS, Sodium dodecyl sulphate | Sigma | #L5750 | For preparing 'TUB' SDS-PAGE gels |
Sonicator | Branson | #101-148-070 | Used for lysing cells grown as adherent cultures. Use 6.5 mm diameter probe, set the sonicator at “Output control” 1, “Duty cycle” 10% and time depending on the cell type used. |
Tabletop centrifuge for 1.5 ml tubes | Eppendorf | 5417R | |
TEMED, N, N, N′, N′-Tetramethylethylenediamine | Sigma | #9281 | |
Trichostatin A (TSA) | Sigma | #T8552 | |
Triton X-100 | Sigma | #T9284 | |
Trizma base (Tris) | Sigma | #T1503 | |
Trypsin | Life Technologies | #15090046 | |
Ultracentrifuge rotors | TLA-55, TLA-100.3 and 70.1 Ti rotors | Set at 4°C or 30°C based on the need of the experiment | |
Ultracentrifuge tubes | Beckman | #357448 | for using with TLA-55 rotor |
Ultracentrifuge tubes | Beckman | #349622 | for using with TLA-100.3 rotor |
Ultracentrifuge tubes | Beckman | #355631 | for using with 70.1 Ti rotor |
Ultracentrifuges | Beckman | Optima L80-XP (or equivalent) and Optima MAX-XP (or equivalent) | Set at 4°C or 30°C based on the need of the experiment |
Vortex mixer | |||
Water bath equipped with floaters or tube holders | Set at 30°C |