Name: single-molecule analysis of sf9 purified superprocessive kinesin-3 family motors
Uploaded: 2022-07-27T13:00:00
Description: Watch this Scientific Journal Video about Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors at JoVE.com

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&#160;fellowship from IIT Gandhinagar.

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 &#176;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...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+motor+toolbox+for+intracellular+transport.">The molecular motor toolbox for intracellular transport.</a> Cell. 112, 467-480 (2003).</li><li>Miki, H., Okada, Y., Hirokawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+kinesin+superfamily:+insights+into+structure+and+function.">Analysis of the kinesin superfamily: insights into structure and function.</a> Trends in Cell Biology. 15 (9), 467-476 (2005).</li><li>Hirokawa, N., Niwa, S., Tanaka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+motors+in+neurons:+transport+mechanisms+and+roles+in+brain+function,+development,+and+disease.">Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease.</a> Neuron. 68 (4), 610-638 (2010).</li><li>Hirokawa, N., Takemura, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+motors+and+mechanisms+of+directional+transport+in+neurons.">Molecular motors and mechanisms of directional transport in neurons.</a> Nature Reviews Neuroscience. 6 (3), 201-214 (2005).</li><li>Patel, N. 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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 successf...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sf9 culture and transfection materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-FLAG M2 affinity</td>
			<td>Biolegend</td>
			<td>651502</td>
			<td>For motility purification</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma</td>
			<td>A6279</td>
			<td>For motility assay and purification</td>
		</tr>
		<tr>
			<td>Cellfectin</td>
			<td>Invitrogen</td>
			<td>10362100</td>
			<td>For Sf9 transfection</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>D5545</td>
			<td>For motility assay mixture</td>
		</tr>
		<tr>
			<td>FLAG peptide</td>
			<td>Sigma</td>
			<td>F3290</td>
			<td>For motility purification</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>For motility purification</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td>Preparing lysing Sf9 cells</td>
		</tr>
		<tr>
			<td>IGEPAL CA 630</td>
			<td>Sigma</td>
			<td>I8896</td>
			<td>Preparing lysing buffer for Sf9 cells</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>For motility purification</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td>For motility assay and purification</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td>For preparing lysis buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>For preparing lysis buffer</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td>For motility purification</td>
		</tr>
		<tr>
			<td>Sf9 cells</td>
			<td>Kind gift from Dr. Thomas Pucadyil (Indian Institute of Science Education and Research, Pune, India).</td>
			<td></td>
			<td>For baculovirs expression and purification</td>
		</tr>
		<tr>
			<td>Sf9 culture bottles</td>
			<td>Thermo Scientific</td>
			<td>4115-0125</td>
			<td>For suspension culture</td>
		</tr>
		<tr>
			<td>Sf-900/SFM medium (1X)</td>
			<td>Thermo Scientific</td>
			<td>10902-096 -500ml</td>
			<td>For culturing Sf9 cells</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S1888</td>
			<td>Preparing lysing buffer for Sf9 cells</td>
		</tr>
		<tr>
			<td>Unsupplemented Grace&#8217;s media</td>
			<td>Thermo Scientific</td>
			<td>11595030 -500ml</td>
			<td>For Sf9 transfection</td>
		</tr>
		<tr>
			<td>Mirotubule Polymerization and Single molecule assay materails</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A2647</td>
			<td>For motility and gliding assay</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td>For blocking motility chamber</td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma</td>
			<td>C9322</td>
			<td>For motility and gliding assay</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D5879</td>
			<td>For dissolving Rhodamine</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>3777</td>
			<td>For preparing buffers</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td>For motility and gliding assay</td>
		</tr>
		<tr>
			<td>Glucose oxidase</td>
			<td>Sigma</td>
			<td>G2133</td>
			<td>For motility and gliding assay</td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>Sigma</td>
			<td>G8877</td>
			<td>For microtubule polymerization</td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma</td>
			<td>P1767</td>
			<td>Preparing PIPES buffer pH 6.9</td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma</td>
			<td>P6757</td>
			<td>For motility and gliding assay</td>
		</tr>
		<tr>
			<td>Microtubule gliding assay materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>26G&#160; needle</td>
			<td>Dispovan</td>
			<td></td>
			<td>For shearing microtubules</td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma</td>
			<td>C3400</td>
			<td>For microtubule glidning assay</td>
		</tr>
		<tr>
			<td>GFP nanobodies</td>
			<td>Gift from Dr. Sivaraj Sivaramakrishnan (University of Minnesota, USA)</td>
			<td></td>
			<td>For attaching motors to the coverslip</td>
		</tr>
		<tr>
			<td>Rhodamine</td>
			<td>Thermo Scientific</td>
			<td>46406</td>
			<td>For preparing labelling tubulin</td>
		</tr>
		<tr>
			<td>Microscope and other instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.5ml, 1.5 and 2-ml microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For Sf9 culture and purification</td>
		</tr>
		<tr>
			<td>10ml&#160; disposable sterile pipettes</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For Sf9 culture and purification</td>
		</tr>
		<tr>
			<td>10ul, 200ul, 1ml micropipette tips</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For Sf9 culture and purification</td>
		</tr>
		<tr>
			<td>15ml concal tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For Sf9 culture and purification</td>
		</tr>
		<tr>
			<td>35mm cell culture dish</td>
			<td>Cole Palmer</td>
			<td>15179-39</td>
			<td>For Sf9 culture</td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Sartorious</td>
			<td></td>
			<td>0.01g-300g</td>
		</tr>
		<tr>
			<td>Benchtop orbial shaking incubator</td>
			<td>REMI</td>
			<td></td>
			<td>For Sf9 suspenculture at 28oC</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>EMCCD Andor iXon Ultra 897</td>
			<td></td>
			<td>For TIRF imaging and acquesition</td>
		</tr>
		<tr>
			<td>Double sided tape</td>
			<td>Scotch</td>
			<td></td>
			<td>For making motility chamber</td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>Fisherfinest</td>
			<td>12-548-5A</td>
			<td>size; 22X30</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Blue Star</td>
			<td></td>
			<td>For making motility chamber</td>
		</tr>
		<tr>
			<td>Heating block</td>
			<td>Neuation</td>
			<td></td>
			<td>Dissolving paraffin wax</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon Eclipse Ti- U</td>
			<td></td>
			<td>To check protein expression</td>
		</tr>
		<tr>
			<td>Lasers</td>
			<td>488nm (100mW)</td>
			<td></td>
			<td>For TIRF imaging</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td>For sample freezing and storage</td>
		</tr>
		<tr>
			<td>Microcapillary loading tip</td>
			<td>Eppendorf</td>
			<td>EP022491920</td>
			<td>For shearing microtubules</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon Eclipse Ti2-E with DIC set up</td>
			<td></td>
			<td>For TIRF imaging</td>
		</tr>
		<tr>
			<td>Mini spin</td>
			<td>Genetix, BiotechAsia Pvt.Ltd</td>
			<td></td>
			<td>For quick spin</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>100X TIRF objective with 1.49NA oil immersion</td>
			<td></td>
			<td>For TIRF imaging</td>
		</tr>
		<tr>
			<td>Optima UltraCentrifuge XE</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>For protein purification</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH-meter</td>
			<td>Corning</td>
			<td>Coring 430</td>
			<td>To adjust pH</td>
		</tr>
		<tr>
			<td>Pipette-boy</td>
			<td>VWR</td>
			<td></td>
			<td>For Sf9 culture and purification</td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 21</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>For protein purification</td>
		</tr>
		<tr>
			<td>Sorvall ST8R centrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Protein purification</td>
		</tr>
		<tr>
			<td>ThermoMixer</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For microtubule polymerization</td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor</td>
			<td>Beckman coulter</td>
			<td></td>
			<td>SW60Ti rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>Beckman</td>
			<td></td>
			<td>5 mL, Open-Top Thinwall Ultra-Clear Tube, 13 x 51mm</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Neuation</td>
			<td></td>
			<td>Sample mixing</td>
		</tr>
		<tr>
			<td>Wax</td>
			<td>Sigma</td>
			<td>V001228</td>
			<td>To seal motility chamber</td>
		</tr>
	</tbody>
</table>

single-molecule analysis of sf9 purified superprocessive kinesin-3 family motors

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&#160;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&#160;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.

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 <strong class=...

Watch this Scientific Journal Video about Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors at JoVE.com

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

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 ...

The protocol offers a simple method for purifying active molecular motors using Sf9 baculovirus system. It also details single molecule motility and multi-motor gliding assays to study the regulatory mechanism of motor proteins. All that traditional vector expression Sf9 baculovirus expression and single step affinity purification, will lead pure and active motor protein to study the mechanistic details of Superprocessive Kinesins in single molecule and multi-motor systems.In addition to Kinesin motors, the protocol can be applied to study biochemical and biophysical properties of other cytoskeletal based proteins such as dionine and myosin. The protocol is detailed and easy to follow. Some suggestions are to handle Sf9 cells carefully as they are prone to contamination, and to follow notes provided in the protocol.Visual demonstration is highly effective, easy to follow and understand the critical steps, especially for those who have never performed these assays. Demonstrating the procedure will be Pushpanjali Soppina and Dipeshwari Shewale, PhD students working with me and Pradeep Naik. To begin, seed the cells in a 35 milliliter dish, at a density of 4.5 times 10 to the fifth cells per milliliter.After 24 hours, mix one microgram of bacmid DNA and 100 microliters of unsupplemented Grace's media in a tube. Mix six microliters of transfection reagent in another tube with 100 microliters of unsupplemented Grace's media. Carefully transfer the content of the first tube into the second tube.Mix them and incubate the mixture for 45 minutes at room temperature. Add 0.8 milliliters of unsupplemented Grace's media to the mixture. Mix and aspirate the Sf900 SFM media from the cells.Add the prepared transfection media drop-wise on the top of the cells and incubate the plate for six hours. Then carefully remove the transfection mixture. Add two milliliters of SF900 SFM media and incubate further at 28 degrees Celsius for 48 hours.Next, check the motor protein expression under an inverted fluorescence microscope. Harvest the media with infected cells, and spin for five minutes, at 500 G.Collect the supernatant and snap freeze the aliquots. Add 10 milliliters of SF900 SFM media with cells and one milliliter of P-zero virus stock, in a sterile, 100 milliliter, conical flask.Incubate at 28 degrees Celsius with constant shaking at 90 rpm. After 72 hours, spin down the cells in a 15 milliliter, sterile conical tube at 500 G for five minutes and collect the supernatant. For large scale protein expression, infect 30 milliliters of the suspension culture with one milliliter of P1 stock virus.72 hours post-infection, check the cells for protein expression and collect the cells in sterile, 50 milliliter, conical tubes. After spinning, discard the supernatant, and collect the cell pellet. To lyse the cells, add three milliliters of ice cold lysis buffer to the cell pellet.Spin the cell lysate at 150, 000 G for 30 minutes, at four degrees Celsius. Collect the supernatant and mix it with 40 microliters of 50%ANTI-FLAG M2 affinity resin. Incubate the mixture at four degrees Celsius for three hours with end-to-end tumbling.Now pellet the FLAG resin by spinning at 500 G for one minute at four degrees Celsius. Wash the FLAG resin pellet three times with ice cold wash buffer. And after the third wash, carefully drain the wash buffer.Next, add 70 microliters of wash buffer containing 100 micrograms per milliliter FLAG peptide, to the resin pellet, and incubate overnight at four degrees Celsius, as demonstrated earlier. After spinning, collect the supernatant containing purified protein into a fresh tube. Prepare a polymerization mix as described in the manuscript, and add 10 microliters of tubulin to the polymerization mixture.Transfer the tube into a heat block, pre-warmed at 37 degrees Celsius, and incubate for 30 minutes. After preparing the microtubule stabilization buffer, warm it, and add it to the polymerized microtubules. Once the motility flow cell chamber is prepared, flow 30 microliters of diluted microtubule solution through the chamber.After 30 minutes, flow 40 to 50 microliters of blocking buffer and incubate it. Prepare a motility mixture and add one microliter of the purified motor to it. Mix well and flow the solution into the motility chamber.Seal both ends of the motility chamber and image under TIRF illumination. Focus on the individual mCitirine tagged motors, moving processively. Mix rhodamine labeled tubulin with unlabeled tubulin in a ratio of one to 10.After 30 minutes of polymerization, gently add 30 microliters of pre-warmed microtubule stabilization buffer and incubate further, for five minutes, at 37 degrees Celsius. Shear the microtubules by pipetting with the capillary loading tip. After preparing the motility flow chamber, flow 50 microliters of Sf9 purified GFP nanobodies and incubate for 30 minutes.Block the cover slip surface by flowing 50 microliters of block buffer. Prepare 50 microliters of the motor mix. Flow it into the chamber after gently mixing the solution and incubate for 30 minutes.Wash the chamber, twice, with 50 microliters of P12-casein. Prepare the gliding assay mixture and mix it gently, before flowing it into the motility chamber. Seal the ends of the motility chamber, and image microtubule gliding under TIRF illumination.After 48 hours of transfection, the untransfected cells appear small, round and firmly attached. However, the transfected cells expressing protein, were loosely attached to the surface and showed enlarged cell diameter. After 72 hours, more than 90%of Sf9 cells expressed fluorescently tagged, kinesin-3 motor KIF1A, and cells were loosely bound to the surface.Kinesin-3 motor protein was purified from the transfected Sf9 cells. The kymograph depicts kinesin-3 motor walking processively, along the microtubule surface. Motility events showed an average velocity and run length of approximately 2.44 micrometers per second and 10.79 micrometers respectively.The kymograph indicates smooth microtubule gliding by the kinesin-3 motors, with an average velocity of approximately 1.37 micrometers per second. The most important thing is to add the stabilization buffer without disturbing the microtubule mix and not to shear the microtubule. The protocol would be useful for other applications, such as ATPase measurements, biophysical analysis, mechanisms, in vitro reconstitution assays, et cetera.Using sf9 purified motors, we've demonstrated recently that kinesin-3 motors are fast and superprocessive. Interestingly, these motors exhibit high ATP turnover rates compared to well-characterized motor, Kinesin-1.

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