This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.
A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.
An immensely crowded cell environment poses many challenges in sorting destined proteins and molecules. This intense workload of organization and spatiotemporal distribution of molecules within the cytoplasm is facilitated by molecular motors and cytoskeletal tracks. Molecular motors are the enzymes that hydrolyze the energy currencies such as ATP and utilize that energy during motion and force generation1. Based on the amino acid sequence similarity, kinesins are grouped into 14 families and despite this similarity, each motor contributes uniquely to the functioning of a cell. Kinesin-3 family motors constitute one of the largest, comprising five subfamilies (KIF1, KIF13, KIF14, KIF16, and KIF28)2, associated with diverse cellular and physiological functions, including vesicle transport, signaling, mitosis, nuclear migration, and development 3,4,5. Impairment in kinesin-3 transport function implicates in many neurodegenerative disorders, developmental defects, and cancer diseases6,7,8,9.
Recent work has demonstrated that kinesin-3 motors are monomers but undergo cargo-induced dimerization and result in fast and superprocessive motility compared to conventional kinesin10,11,12,13. Their biochemical and biophysical characterization needs a large quantity of purified, active proteins. However, their production in the prokaryotic expression system resulted in inactive or aggregated motors, presumably due to incompatible protein synthesis, folding and modification machinery14,15,16,17,18. To circumvent such limitations and increase the yield, here we have established a robust Sf9-baculovirus expression system to express and purify these motors.
The baculovirus expression system uses Sf9 insect cell lines as a host system for high-throughput eukaryotic recombinant protein expression19,20. Baculovirus possesses a strong polyhedrin promoter that assists in heterologous gene expression and the production of soluble recombinant proteins17. Due to its cost-effectiveness, safe to handle and high amount of active protein expression, it has become a powerful tool21. To express a protein of interest, a key step is to generate a recombinant bacmid. Since the commercially available bacmid generating kits are expensive and we will be working with more samples, we developed an in-house protocol for both large and small inserts of kinesin-3 motors into bacmids. Sf9-purified kinesin-3 motors were used to characterize in vitro single-molecule and multi-motor microtubule gliding properties using total internal reflection fluorescence (TIRF) microscopy. Motors are C-terminally tagged with 3-tandem fluorescent molecules (3xmCit) to provide enhanced signal and decreased photobleaching. Due to its increased signal-to-noise ratio, less phototoxicity, and selective imaging of a very small area close to the coverslip, TIRF imaging has been widely used to visualize protein dynamics at the single-molecule level in vivo and in vitro.
This study discusses the purification of kinesin-3 motors by employing Sf9-baculovirus expression system and in vitro single-molecule imaging and multi-motor gliding analysis of motors using TIRF microscopy. Altogether, this study shows that the motility properties of Sf9 purified motors are identical to that of motors prepared from mammalian cell lysates. Hence, we believe that the Sf9-baculovirus system can be adapted to express and purify any motor protein of interest.
1. Sf9 culture, transfection, and virus generation
NOTE: Maintain Sf9 cells in 30 mL of Sf-900/SFM medium in 100 mL sterile, disposable conical flask without any antibiotic/antimycotic at 28 °C. Keep the suspension culture in an orbital shaker at 90 rpm. Supply of CO2 and humidity maintenance is not required. Cells are usually subcultured every fourth day by inoculating 0.5 x 106 cells/mL to reach 2.0 x 106 cells/mL density on the fourth day.
2. Sf9 purification of kinesin-3 motors
3. In vitro single-molecule motility assay using Sf9-purified kinesin-3 motors
NOTE: The Sf9-purified kinesin-3 motors can be used to study biochemical and biophysical properties such as ATP turnover rate, microtubule affinity, velocity, run length, step size, and force generation. Here, a detailed protocol for in vitro single-molecule motility analysis of KIF1A(1-393LZ) using Total Internal Reflection Fluorescence (TIRF) microscopy is described. For all the buffers composition and reagents, please refer to Supplemental Table 1.
4. In vitro microtubule gliding assay
NOTE: To understand the collective behavior of kinesin-3 motors, in vitro microtubule gliding assay was performed18,30,31. Where motors are immobilized onto the coverslip in an inverted position and upon adding microtubules into the chamber, microtubules land on motors and glide along as the motors try to walk on them (Figure 5A,B).
To express and purify active and functional recombinant motor proteins at a large scale using the Sf9-baculovirus expression, the system needs generation of viral particles stably carrying a coding sequence to infect Sf9 cells. To achieve this, Sf9 cells were transfected with recombinant bacmid encoding KIF1A(1-393LZ)-3xmCit-FLAG. After 72 h, a significant population of cells showed expression of green fluorescent protein (mCitrine) with enlarged cells and nuclei (Figure 1 and Figure 2), suggesting successful generation of viral particles (P0). These viral particles were further expanded by infecting fresh Sf9 population at a higher volume to generate P1 viral stocks for storage and large-scale infection. For purification of kinesin-3 motor, a 30 mL culture of Sf9 cells were infected with 1 mL of P1 viral particles. After 72 h of infection, cells brightly expressing green fluorescent protein were harvested, lysed, and purified using C-terminal FLAG tag affinity purification with anti-FLAG antibody-coated resin. Interestingly, the addition of FLAG peptide to the beads attached with kinesin-3 motors eluted most of the protein in a single fraction and the purified protein was of high purity, shown as a band of ~120 kDa in Lane 7 of Figure 3.
The successful polymerization of long and healthy microtubules to study the motility properties of S9-purified kinesin-3 motors at a single-molecule level requires pure tubulin. In the present study, the microtubules were polymerized using 2X cycled tubulin purified from the goat brain at a critical concentration between ~2.5-3.0 mg/mL for 30 min in the absence of taxol, as taxol decreases the critical concentration of microtubule nucleation and generates a large number of short microtubules33,34,35. Taxol was added post-polymerization to stabilize the microtubules and prevent depolymerization. Microtubules polymerized using this method resulted in long and uniform structures (Video 1) with an average length of 60-70 µm.
In vitro single-molecule motility analysis of Sf9-purified kinesin-3 motor, KIF1A(1-393LZ) under TIRF illumination (Figure 4B) exhibited unidirectional, fast, and superprocessive motion along the microtubule (Video 2), as shown in the kymograph as long diagonal white lines (Figure 4C). Tracking analysis of these long superprocessive motility events showed an average velocity and run length of 2.44 ± 0.02 µms-1 and 10.79 ± 0.28 µm (Figure 4D–E), respectively. These results are in agreement with our previously published data12,13 using motors prepared from mammalian cell lysate by overexpression. Furthermore, the multi-motor microtubule-gliding assay using Sf9-purified motors attached on the coverslip under TIRF illumination exhibited long, uniform, and unidirectional motion (Figure 5C and Video 3). Tracking analysis of these long microtubule gliding events showed an average velocity of 1.37 ± 0.01 µms-1 (Figure 5D).
Together, these results suggest that the Sf9-baculovirus system provides an excellent system for the expression and purification of active motor proteins that are generally difficult using the prokaryotic system. FLAG tag offers an advantage of the single-step purification of protein in its pure form. Furthermore, microtubule polymerization using fresh and pure tubulin and their stabilization after the polymerization yields long and uniform microtubules. Additionally, in vitro single-molecule motility analysis suggests that Sf9-purified motors were robust and exhibited motility properties similar to motors expressed in mammalian systems.
Figure 1: 48 h post-transfection of Sf9 cells with fluorescently-tagged kinesin-3 motor: Sf9 cells were plated on a 35 mm dish at a density of 4.5 x 105 cells/mL. After 24 h, cells were transfected with recombinant bacmid DNA encoding specific kinesin-3 motor, KIF1A(1-393LZ)-3xmCit-FLAG using transfection reagent. After 48 h of transfection, images were captured under differential interference and contrast (DIC) and Epifluorescence illumination. (A) Untransfected cells, small, round, and firmly attached. (B) Transfected cells expressing KIF1A(1-393LZ)-3xmCit-FLAG tagged protein showing enlarged cell diameter and loosely attached to the surface. Scale bar, 100 µm. (C) Bar graph indicating average cell diameter of untransfected and transfected cells. Error bars represent mean ± SD. N = 50 cells. Student's t-test is used to find the significance value (****p < 0.0001). Please click here to view a larger version of this figure.
Figure 2: 72 h post-transfection of Sf9 cells with fluorescently-tagged kinesin-3 motor: Images showing more than 90% of Sf9 cells expressing fluorescently-tagged kinesin-3 motor, KIF1A(1-393LZ). Cells are loosely bound to the surface. Please click here to view a larger version of this figure.
Figure 3: Sf9-purified kinesin-3 motor: Coomassie-stained SDS-PAGE gel representing Sf9-purified kinesin-3 motor, KIF1A(1-393LZ) C-terminally tagged with 3xmCit-FLAG. From left to right, Protein ladder (M), BSA standard protein control, 0.2 µg (lane 2), 0.4 µg (lane 3), 0.6 µg (lane 4), 0.8 µg (lane 5), and 1.0 µg (lane 6), purified kinesin-3 motor (S) (lane 7). BSA was used as a standard to estimate purified protein concentration. Please click here to view a larger version of this figure.
Figure 4: In vitro single-molecule motility assay using Sf9 purified kinesin-3 motors:(A) Schematic flow cell motility chamber prepared using a glass slide, double-sided tape, and glass coverslip. (B) Cartoon diagram illustrating in vitro single-molecule imaging of fluorescently tagged motors moving along the microtubule surface adsorbed onto the coverslip using Total Internal Reflection Fluorescence (TIRF) microscopy. Only the molecules very close to the coverslip get excited due to the evanescent field (~150-200 nm) formed by the complete reflection of incident light when illuminated at an angle beyond the critical angle. (C) Kymograph depicts kinesin-3 motor walking processively (diagonal white lines) along the microtubule surface. Time on the y-axis (vertical arrow) and distance on the x-axis (horizontal arrow). (D–E) The histograms of velocity (D) and run length (E) were determined by tracking individual motors walking on the microtubule surface frame-by-frame and histograms were plotted for each population of motors and fit to a single Gaussian. The peak represents the average velocity and run length and values are shown on the graph as mean ± SEM. N = Number of events counted. Please click here to view a larger version of this figure.
Figure 5: In vitro microtubule gliding assay using Sf9 purified kinesin-3 motors: (A) Schematic of flow cell chamber prepared using a glass slide, double-sided tape, and glass coverslip. (B) Cartoon diagram illustrating rhodamine-labeled microtubules gliding on the surface of constitutively active kinesin-3 motors. As kinesin-3 motors are C-terminally tagged with mCitrine, GFP-nanobodies were used to attach them to the glass surface. Subsequently, sheared rhodamine-labeled microtubules were introduced into the flow chamber and images of microtubule gliding movement were captured under TIRF illumination. (C) The kymograph represents smooth microtubule gliding (diagonal white stripe) by the kinesin-3 motors. Time on the y-axis (vertical arrow) and distance on the x-axis (horizontal arrow). D. Histogram of the velocity were plotted for a population of microtubules and fit to a single Gaussian peak. Average gliding velocity is indicated on the top-left corner as mean ± SEM. N = Number of molecules counted. Please click here to view a larger version of this figure.
Video 1: Polymerized microtubules. TIRF imaging of polymerized microtubules before diluting for motility assay. The movie was acquired at 100 ms exposure and the video is played at 60 frames/s. Scale bar, 10 µm. Please click here to download this Video.
Video 2: In vitro single-molecule assay of the constitutively active kinesin-3 motor. Fluorescently-tagged KIF1A(1-393LZ) motors purified from Sf9 cells progressively moving along the microtubule surface (unlabeled) adsorbed on the coverslip in a motility assay chamber. Imaging was performed in TIRF illumination and images were acquired at 100 ms exposure. Video is played at 30 frames/s. Scale bar, 10 µm. Please click here to download this Video.
Video 3: Microtubule gliding assay of the constitutively active kinesin-3 motor. Rhodamine-labeled microtubules smoothly gliding on the surface of the glass coverslip coated with Sf9 purified KIF1A(1-393LZ) motor. Imaging was performed in TIRF illumination and images were acquired at 100 ms exposure. Video is played at 60 frames/s. Scale bar, 10 µm. Please click here to download this Video.
Supplemental Table 1: Buffers and reagents composition. Please click here to download this File.
The Sf9-baculovirus expression system is one of the most versatile and successful methods for high-throughput protein production19,36,37. The posttranslational modification ability of Sf9 cells is highly comparable to the mammalian system15. A considerable disadvantage of using this system is that it is slow and sensitive to contamination. One of the most critical steps is efficient infection and successful expression in Sf9 cells, which requires proper handling and maintenance of Sf9 cells without any contamination. The system requires dedicated dust-free space and instruments such as a temperature-controlled incubator and shaker. Irregular cell culture practice and use of overgrown culture will result in poor infection, slow growth, and significant cell death.
The FLAG tag purification of protein expressed in Sf9 cells has several advantages. First, Sf9 cells can be lysed efficiently to get most of the protein in its active and soluble form. Secondly, FLAG resin has less nonspecific protein adsorption than HIS and GST resin38. As a result, a significant amount of the protein gets released in a single-step elution in its pure form. The Sf9-purified kinesin-3 motors showed robust microtubule-stimulated ATP turnover rates in the biochemical ATPase assays14. Similarly, in in vitro single-molecule motility assays, these motors showed fast and superprocessive motility on the surface of the microtubule. Interestingly, these motility properties showed remarkable similarity with the motility properties measured using motors prepared from mammalian cell lysates11,12,13. Additionally, the measured stepping rate of kinesin-3 motors in the in vitro single-molecule motility assay closely matches the rate of ATP hydrolysis measured in biochemical ATPase assay, suggesting motors hydrolyze one ATP molecule per step14. Together, these results suggest that kinesin3 motors purified from the Sf9-baculovirus expression system yielded a significantly large population of active and soluble proteins. Therefore, we believe that the Sf9-baculovirus system can be employed to purify motor proteins of interest with necessary modifications.
In vitro single-molecule motility assay of kinesin-3 motors that exhibit superprocessive motility needs polymerization of long polymerized microtubules. A critical step in in vitro polymerization of long microtubules is to prepare a polymerization mixture with critical tubulin concentration, incubation time, and appropriate buffer pH. The tubulin used for microtubule polymerization should be highly competent, pure, and stored in liquid nitrogen in small aliquots to avoid freezing and thawing. Tubulin, once thawed, should not be frozen back for future polymerization because tubulin loses its competency significantly in every freeze-thaw cycle. Incompetent tubulin will not contribute to the microtubule polymerization but will hinder the polymerization process and result in short microtubules with unknown defects. The optimal tubulin concentration in the polymerization mixture should be between 2.5-3 mg/mL and the mixture should be kept on ice for 5 min before incubating it at 37 °C for polymerization. The duration of microtubules polymerization should be between 20-30 min and should not exceed 30 min, as prolonged polymerization often results in the formation of short and fragmented microtubules.
Next, stabilization of polymerized microtubules is a second critical step. Taxol is poorly soluble in water and precipitates in an aqueous solution39. Therefore, prepare the stabilization buffer freshly with 10 µM taxol, add gently over the polymerized microtubules mixture, and incubate further without disturbing. Always use frozen taxol aliquot from the -20 °C freezer to prepare the stabilization buffer, as thawed aliquot results in the precipitation/aggregation of polymerized microtubules. Always handle the polymerized microtubules using beveled tips to avoid breakage. Microtubules polymerized using this method yield long microtubules with 60-70 µm long suitable for superprocessive motility analysis. The next critical step is the stable adsorption of microtubules to the coverslip surface in the motility chamber. To achieve this, coverslips should be stored in a dry place because exposure of coverslips to the humid environment significantly decreases the microtubule adsorption efficiency and their stability.
Preparing a motility assay mixture with proper ingredients at particular concentrations and optimal motor protein concentration is important to achieve robust motility events along the microtubule surface. The most crucial component in generating smooth and processive motility is using a stable ATP solution in the assay mixture. (Thus, care should be taken while preparing the ATP stock. Dissolving the sodium salt of ATP in 10 mM PIPES buffer pH 6.9, yields a clear solution with a very low pH. ATP is extremely unstable at low pH and rapidly hydrolyzes to ADP and phosphate, thus keeping the solution cold and immediately adjusting the pH to 7.0 using concentrated KOH prevents hydrolysis.) Flow the motility mixture into the flow cell and seal both the edges with liquid paraffin to prevent liquid flow and drying of the chamber while imaging under TIRF illumination.
As kinesin-3 motors are fast and superprocessive along the microtubule tracks, their imaging, data acquisition and analysis pose several challenges. Single-molecule imaging and data acquisition should be performed under low laser intensity to prevent photobleaching of fluorescent proteins before the motor completes its processive run. The acquired data would be of low intensity with poor signal-to-noise ratio. Consequently, tracking of these individual motility events using existing automated tracking software to characterize their motility events would be difficult. Therefore, data analysis has to be performed manually by accurately tracking fluorescently tagged motor spots moving along the microtubule tracks frame-by-frame with minimal errors. For data analysis, preferentially, choose the motility events on the long microtubule tracks to avoid motors reaching the microtubule end before completing their run. For consistent and reproducible results, consider only those motility events for tracking, at least, the last 10 frames (500 ms) with a clear starting and ending of the processive run.
Overall, the method offers several advantages over the existing method, such as simple and one-step purification of active motor proteins, preparation of long microtubules, and detailed step-by-step protocol with instructions to achieve good motility. In principle, the method can be applied to express and purify any class and type of proteins, including but not limited to myosin, kinesin, and dynein. Indeed, the baculovirus expression system has been successfully established to purify several viral vaccines, gene therapy vectors and other pharmaceutical proteins20.
The authors have nothing to disclose.
V.S. and P.S. thank Prof. Kristen J. Verhey (University of Michigan, Ann Arbor, MI, USA) and Prof. Roop Mallik (Indian Institute of Technology Bombay (IITB), Mumbai, India) for their unconditional support throughout the study. P.S. thanks Dr. Sivapriya Kirubakaran for her support throughout the project. V.S. acknowledges funding through DBT (Grant No.: BT/PR15214/BRB/10/1449/2015 and BT/RLF/Re-entry/45/2015) and DST-SERB (Grant No.: ECR/2016/000913). P.K.N acknowledges ICMR for funding (Grant No. 5/13/13/2019/NCD-III). P.S. acknowledges funding from DST (Grant No.: SR/WOS-A/LS-73/2017). D.J.S acknowledges fellowship from IIT Gandhinagar.
Sf9 culture and transfection materials | |||
anti-FLAG M2 affinity | Biolegend | 651502 | For motility purification |
Aprotinin | Sigma | A6279 | For motility assay and purification |
Cellfectin | Invitrogen | 10362100 | For Sf9 transfection |
DTT | Sigma | D5545 | For motility assay mixture |
FLAG peptide | Sigma | F3290 | For motility purification |
Glycerol | Sigma | G5516 | For motility purification |
HEPES | Sigma | H3375 | Preparing lysing Sf9 cells |
IGEPAL CA 630 | Sigma | I8896 | Preparing lysing buffer for Sf9 cells |
KCl | Sigma | P9541 | For motility purification |
Leupeptin | Sigma | L2884 | For motility assay and purification |
MgCl2 | Sigma | M2670 | For preparing lysis buffer |
NaCl | Sigma | S7653 | For preparing lysis buffer |
PMSF | Sigma | P7626 | For motility purification |
Sf9 cells | Kind gift from Dr. Thomas Pucadyil (Indian Institute of Science Education and Research, Pune, India). | For baculovirs expression and purification | |
Sf9 culture bottles | Thermo Scientific | 4115-0125 | For suspension culture |
Sf-900/SFM medium (1X) | Thermo Scientific | 10902-096 -500ml | For culturing Sf9 cells |
Sucrose | Sigma | S1888 | Preparing lysing buffer for Sf9 cells |
Unsupplemented Grace’s media | Thermo Scientific | 11595030 -500ml | For Sf9 transfection |
Mirotubule Polymerization and Single molecule assay materails | |||
ATP | Sigma | A2647 | For motility and gliding assay |
BSA | Sigma | A2153 | For blocking motility chamber |
Catalase | Sigma | C9322 | For motility and gliding assay |
DMSO | Sigma | D5879 | For dissolving Rhodamine |
EGTA | Sigma | 3777 | For preparing buffers |
Glucose | Sigma | G7021 | For motility and gliding assay |
Glucose oxidase | Sigma | G2133 | For motility and gliding assay |
GTP | Sigma | G8877 | For microtubule polymerization |
KOH | Sigma | P1767 | Preparing PIPES buffer pH 6.9 |
PIPES | Sigma | P6757 | For motility and gliding assay |
Microtubule gliding assay materials | |||
26G needle | Dispovan | For shearing microtubules | |
Casein | Sigma | C3400 | For microtubule glidning assay |
GFP nanobodies | Gift from Dr. Sivaraj Sivaramakrishnan (University of Minnesota, USA) | For attaching motors to the coverslip | |
Rhodamine | Thermo Scientific | 46406 | For preparing labelling tubulin |
Microscope and other instruments | |||
0.5ml, 1.5 and 2-ml microcentrifuge tubes | Eppendorf | For Sf9 culture and purification | |
10ml disposable sterile pipettes | Eppendorf | For Sf9 culture and purification | |
10ul, 200ul, 1ml micropipette tips | Eppendorf | For Sf9 culture and purification | |
15ml concal tubes | Eppendorf | For Sf9 culture and purification | |
35mm cell culture dish | Cole Palmer | 15179-39 | For Sf9 culture |
Balance | Sartorious | 0.01g-300g | |
Benchtop orbial shaking incubator | REMI | For Sf9 suspenculture at 28oC | |
Camera | EMCCD Andor iXon Ultra 897 | For TIRF imaging and acquesition | |
Double sided tape | Scotch | For making motility chamber | |
Glass coverslip | Fisherfinest | 12-548-5A | size; 22X30 |
Glass slide | Blue Star | For making motility chamber | |
Heating block | Neuation | Dissolving paraffin wax | |
Inverted microscope | Nikon Eclipse Ti- U | To check protein expression | |
Lasers | 488nm (100mW) | For TIRF imaging | |
Liquid nitrogen | For sample freezing and storage | ||
Microcapillary loading tip | Eppendorf | EP022491920 | For shearing microtubules |
Microscope | Nikon Eclipse Ti2-E with DIC set up | For TIRF imaging | |
Mini spin | Genetix, BiotechAsia Pvt.Ltd | For quick spin | |
Objective | 100X TIRF objective with 1.49NA oil immersion | For TIRF imaging | |
Optima UltraCentrifuge XE | Beckman Coulter | For protein purification | |
Parafilm | Eppendorf | ||
pH-meter | Corning | Coring 430 | To adjust pH |
Pipette-boy | VWR | For Sf9 culture and purification | |
Sorvall Legend Micro 21 | Thermo Scientific | For protein purification | |
Sorvall ST8R centrifuge | Thermo Scientific | Protein purification | |
ThermoMixer | Eppendorf | For microtubule polymerization | |
Ultracentrifuge rotor | Beckman coulter | SW60Ti rotor | |
Ultracentrifuge tubes | Beckman | 5 mL, Open-Top Thinwall Ultra-Clear Tube, 13 x 51mm | |
Vortex mixer | Neuation | Sample mixing | |
Wax | Sigma | V001228 | To seal motility chamber |