Special thanks to Kris Ngai and Jason Del Rio for their support and assistance in this project. This work was supported by RO1 grant GM070676 to SPG.

Strains of flies can be purchased and amplified in the lab. Depending on the amount of Drosophila culture vials received, one need to frequently 'flip' the culture vials, once the vials has reached maximum capacity. The amplification continues until approximately 50 Drosophila culture vials are filled. One then makes 'fly cups' with 100 ml tricorn beakers (Fly cup making is discussed in the next section). After 24 hours, use embryos from these cups to seed new Drosophila culture vials with affixed fly food at the bottom. At room temperature, the growth time for Drosophila embryos from overnight embryos to full grown flies are about 8.5 days. Seeded embryos hatch after 12-15 hours into the first larvae stage. The larvae grow for about 4 days molting twice into the second and third larvae stage, at 24 and 48 hours after hatching. The larvae then encapsulate into pupariums and submit to a 4 day metamorphosis until emerging from their pupal cases 1. Allow for more growth for about two to three days in the Drosophila culture vials to increase the amount of flies in each. When 400 vials are filled, there will be a sufficient amount of flies for fifty egg laying cups (About 1,000 female flies per cup). Flies need time to adjust to their new environment. It usually takes about two days for them to be ready for collection, after being transferred to the cups. Replacing agar plates daily will keep the fly cups clean, which allow flies to live longer. (For further understanding of Drosophila care and amplification, please refer to D. B. Roberts' Drosophila: A Practical Approach 2).
A good source to obtain large amounts of Drosophila embryos are population cages 3, which are commercially available. The assembly and maintenance of population cages can be seen on chapter 5 and 7 in the book Drosophila Melanogaster: Practical Uses in Cell and Molecular Biology4. However, population cages are expensive and are hard to maintain. Since population cages can hold an abundant amount of flies, the use of carbon dioxide is necessary for transferring and feeding flies. As stated before, carbon dioxide decreases the health of the flies, causing them not to lay as well. On a small scale, 100 ml tricorn beakers can be used. With fifty 100 ml beakers, 9 grams of dechorionated embryos may be attained. In order remove the dead flies, unused yeast paste and other impurities we have created a double layered sieve catcher with different mesh sizes. One sieve traps unwanted materials like dead flies while allowing embryos to pass through (350 micron pore size). The second part contains a mesh (120 microns pore size), which is meant to catch Drosophila embryos and other impurities will pass through the mesh. 
1. Fly Cup Preparation and Embryo Collection
<ol>
 <li> Flip the amplified flies from multiple vials into an empty plastic vial, until the amount of flies have reached one inch of the vial's height. Then, transfer those flies into a 100 ml egg collection cup (tricon beaker with nylon mesh windows). Keep tapping the cup on a hard surface to prevent the escape of flies when transferring flies. Cover cup with agar plate containing yeast paste and allow 24-48 hrs of stabilization time.</li>
 <li> Collect overnight agar plates from fifty fly cups and wash the contents of the plates to the sieve catcher using a clean paint brush and running water. Flesh the embryos in the sieve with plenty of water until all the yeast paste is washed away. </li>
 <li> Dechorionate the cleaned embryos by immersing them in 50% bleach for 3 min. </li>
 <li> Rinse with distilled water extensively until embryos loose the bleach odor. Then dry the mesh with embryos by placing it on a c fold towel multiple times. Transfer the embryos to a clean vial and record the weight. </li>
</ol>
2. Embryo Homogenization and Clarification
<ol>
 <li> Place dechorionated embryos in Dounce homogenizer with 1.5 x volume of ice-cold extraction buffer. </li>
 <li> Do 5 strokes with the loose pestle. Aliquot the homogenate into clean centrifuge tubes. Centrifuge at 15,000 g for 40 min. at 4 &deg;C. </li>
 <li> Carefully collect the clear supernatant liquid without the upper white lipid layer or the lower pellet. </li>
 <li> Transfer supernatant to clean centrifuge tubes and centrifuge at 50,000 g for 30 min. at 4 &deg;C</li>
 <li> Resulting high-speed supernatant collected as explained above can be used immediately or can be snap frozen and stored at -80 &deg;C for months. </li>
</ol>
3. Microtubule Polymerization and Kinesin Binding
<ol>
 <li> Thaw the frozen high-speed supernatant to room temperature. To polymerize microtubules, add GTP (0.3 mM) and taxol (20 &mu;M) and agitate gently at room temperature for 20 min in a rotating shaker. </li>
 <li> To bind kinesin, add 2.5 mM nonhydrolyzable ATP analog 5'-adenylyl imidodiphosphate (AMPPNP), followed by a 10 min agitation at room temperature in a rotating shaker. </li>
</ol>
4. Differential Sedimentation of Microtubules and Kinesin
<ol>
 <li> Sediment through an equal volume sucrose cushion (20% sucrose and 10 &mu;M taxol in extraction buffer) by centrifugation at 23,000 g (sw41ti rotor, TLS-55) for 30 min. at 4 &deg;C. </li>
 <li> Wash pellet by resuspension in 10% of original homogenate volume in extraction buffer containing 10 &mu;M taxol and 75 mM NaCl. </li>
 <li> Repeat the sedimentation with another equal volume sucrose cushion as in step 4.1. </li>
 <li> Re-suspend pellet from salt wash in 5% extraction buffer with 20 &mu;M taxol, 75 mM NaCl, 10 mM MgSO4, and 10 mM ATP. </li>
 <li> Sediment at 120,000 g (sw41ti rotor: 31,000 rpm, TLS-55: 42,000 rpm) for 20 mins at 4 &deg;C. Collect supernatant (kinesin fraction). </li>
 <li> Perform a centrifugal filtration at 14,000 g for 15 min at 4 &deg;C. Recover concentrated sample and repeat filtration with a new filter as above for 30 minutes and collect the concentrated sample. </li>
 <li> Perform a protein assay to determine the concentration. Briefly, varying concentrations of a known protein (bovine serum albumin; BSA) was mixed with a known volume of Bradford reagent and measured the optical density at wavelength 595nm. Then an optical density versus concentration standard curve was established to calculate the unknown concentration of purified kinesin fraction using the optical density of this sample measured under similar conditions as that of the standard BSA samples. Gel electrophoresis as well as western blotting was also performed using a known amount of the purified kinesin. </li>
 <li> Aliquot kinesin fraction into small volumes of desired concentration and snap freeze in liquid nitrogen to store at -80 &deg;C. </li>
</ol>
5. Representative Results
Full-length functional Kinesin was purified from Drosophila embryos. Figure 1 depicts the silver stained gel showing the different fractions during purification. Lane 1 is a sample of the high speed supernatant after clarification (step 2.5), lane 2 is a sample of the pellet after clarification, lane 3 is a sample of the supernatant after the first sedimentation with sucrose cushion (step 4.1), lane 4 is a sample of the pellet after the first sedimentation, lane 5 is a sample of the supernatant after the second sedimentation (step 4.3), lane 6 is a sample of the pellet after the second sedimentation, lane 7 is a sample of the pellet of the final sedimentation (step 4.5), lane 8 is the purified kinesin sample, lane 9 is the filtered kinesin sample, and lane 10 is the marker. The concentrated band in lane 8 and 9 at around 115 kD is kinesin heavy-chain 1, which is consistent with Figure 1, lane 8 of the Saxton paper published in 19885. Figure 2 represents the c western blot of the purified kinesin fraction detected using the kinesin heavy chain antibody. The antibody AKINO1-A does not significantly cross-react with other kinesin family members 6. Note that although this protocol results in a good source of functional kinesin, while the fraction is enriched for kinesin-1, there are likely contaminants, including potentially other kinesins and other motor proteins. We removed a variety of smaller contaminants by filtration. As far as removing other motors which cannot be done by size alone, the purification is similar to what was done by others to study kinesin 7, and we believe that the majority of the active motor is Kinesin-1, but more sophisticated purification would allow removal of potential motor contaminants, such as was done by Cole et al8 and Saxton et al.9 
The processivity of kinesin was evaluated by in vitro single molecule microtubule binding assay, as described in more detail in the book by Scholey entitled "Motility Assay for Motor Proteins"10. Briefly, polystyrene beads with a single active motor were brought into contact with the microtubule, in the presence of saturating (1 mm) ATP. The motor attached to the microtubule, and started walking away from the center of the laser trap. At a pre-defined displacement of the bead from the trap center (100 nm) the laser beam power was automatically turned off, allowing the motor to walk along the MT under no load. The movie shows a 500 nm diameter bead with single kinesin walking along MT. The length of the video screen corresponds to 20 &mu;m. Figure 3 represents the measured distribution of run-lengths for single full-length Drosophila kinesin molecules, purified according to the protocol presented here. The exponential fit to the distribution provides the average runlength of single kinesin 1.55 &plusmn; 0.1 &mu;m and 1.28 &plusmn; 0.12 &mu;m for the filtered and unfiltered sample respectively. 
<img src="/files/ftp_upload/3501/3501fig1.jpg" alt="Figure 1" /> 
Figure 1. Silver stained gel of the purified fractions: Samples from each fraction were run in a 10% gel. 10 &mu;g of protein was loaded in each lane. Lane 1 is a sample of the high speed supernatant after clarification (step 2.5), lane 2 is a sample of the pellet after clarification, lane 3 is a sample of the supernatant after the first sedimentation with sucrose cushion (step 4.1), lane 4 is a sample of the pellet after the first sedimentation, lane 5 is a sample of the supernatant after the second sedimentation (step 4.3), lane 6 is a sample of the pellet after the second sedimentation, lane 7 is a sample of the pellet of the final sedimentation (step 4.5), lane 8 is the purified kinesin sample, lane 9 is the filtered kinesin using a 100 kD cut-off Amicon ultra 0.5 ml centrifugal filter (Millipore, USA). The blue arrows indicate the proteins whose amounts were decreased and the red arrow indicate the concentration of kinesin due to the centrifugal filtration step used in this protocol. 
<img src="/files/ftp_upload/3501/3501fig2.jpg" alt="Figure 2" /> 
Figure 2. Purified KHC fraction blotted against anti-kinesin antibody: Purified kinesin sample was subjected to gel electrophoresis and blotted (in nitrocellulose membrane) against primary antibody AKINO1-A (1:1,000 in TBST ) for 1 hour at room temperature followed by incubation in Donkey &#x2013;anti-rabbit antibody (1:10,000 in TBST) for an hour at room temperature. An ECL (chemiluminescent) kit was used to detect the kinesin signal shown above. 
<img src="/files/ftp_upload/3501/3501fig3.jpg" alt="Figure 3" /> 
Figure 3. Runlength and Velocity of single Drosophila full length kinesin: (A) and (B) Runlength and Velocity of filtered kinesin sample. (C) and (D) Runlength and velocity of unfiltered kinesin sample. 
Figure 4. Video of Kinesin motility. <a href="https://www.jove.com/files/ftp_upload/3501/3501movie4.avi" target="_blank">Click here to view video</a>.

<ol>
	<li>Ashburner, M., Thompson, J. N., Ashburner, M., Wright, T. R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+laboratory+culture+of+Drosophila+2A.">The laboratory culture of Drosophila 2A.</a> The genetics and biology of Drosophila. , 1-81 (1978).</li><li>Roberts, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A Practical Approach. , (1998).</li><li>Kunert, N., Brehm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+production+of+Drosophila+Embryosand+Chromatographic+Purification+of+Native+Protein+Complexes.">Mass production of Drosophila Embryosand Chromatographic Purification of Native Protein Complexes.</a> Methods in Molecular Biology. 420, 359 (2008).</li><li>Goldstein, L. S. B., Fyrberg, E. A., Wilson, L., Matsudaira, P. T., Eds, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster:+Practical+Uses+in+Cell+and+Molecular+Biology.">Drosophila melanogaster: Practical Uses in Cell and Molecular Biology.</a> Methods in Cell Biology. 44, (1994).</li><li>Saxton, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+kinesin:+Characterization+of+microtubule+motility+and+ATPase.">Drosophila kinesin: Characterization of microtubule motility and ATPase.</a> Proceedings of the National Academy of Science. 85, 1109 (1988).</li><li>Shubeita, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+motor+copy+number+on+the+intracellular+transport+of+kinesin-1-driven+lipid+droplets.">Consequences of motor copy number on the intracellular transport of kinesin-1-driven lipid droplets.</a> Cell. 135, 1098-1098 (2008).</li><li>Svoboda, K., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+and+Velocity+Measured+for+Single+Kinesin+Molecules.">Force and Velocity Measured for Single Kinesin Molecules.</a> Cell. 77, 773 (1994).</li><li>Cole, D. G., Saxton, W. M., Sheehan, K. B., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+"Slow"+Homotetrameric+Kinesin-related+Motor+Protein+Purified+from+Drosophila+embryos.">A "Slow" Homotetrameric Kinesin-related Motor Protein Purified from Drosophila embryos.</a> J. Biol. Chem. 269, 22917-22917 (1994).</li><li>Saxton, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Analysis+of+Microtuble+Motor+Proteins.">Isolation and Analysis of Microtuble Motor Proteins.</a> Drosophila Melanogaster. Bloomington: Academic. 44, 279 (1994).</li><li>Scholey, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+Assay+for+Motor+Proteins.">Motility Assay for Motor Proteins.</a> Methods in Cell Biology. 39, 138 (1993).</li><li>Wagner, M. C., Pfister, K., Brody, S., Bloom, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+kinesin+from+bovine+brain+and+assay+of+microtubule-stimulated+ATPase+activity.">Purification of kinesin from bovine brain and assay of microtubule-stimulated ATPase activity.</a> Methods in Enzymology. 196, 157 (1991).</li><li>Aizawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin+Family+in+Murine+Nerwous+System.">Kinesin Family in Murine Nerwous System.</a> The Journal of Cell Biology. 119, 1287-1287 (1992).</li><li>Ori-McKenney, K. M., Xu, J., Gross, S. P., Vallee, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cytoplasmic+dynein+tail+mutation+impairs+motor+processivity.">A cytoplasmic dynein tail mutation impairs motor processivity.</a> Nature Cell Biology. 12, 1228-1228 (2010).</li></ol>

We have nothing to disclose.

Bovine brain is the most widely used starting material 11 to purify full-length kinesin, though murine brain has been used as well 12. A large disadvantage of using bovine brain as a kinesin source is the availability of fresh starting material: slaughterhouses are typically inaccessible, and the brain must be extremely fresh in order to obtain active kinesin. Further, only the brains of young cows are effective. Finally, genetic manipulation of cows is currently not a viable option, so only 'wild-t...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent or material</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 	<td>Agar (Granulated)</td>
 <td>Fisher</td>
 <td>1423-500</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>Dextrose (D-Glucose) Anhydrous</td>
 <td>Fisher</td>
 <td>D16-3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Petri Dishes</td>
 <td>Becton</td>
 <td>35 1007</td>
 <td>60x15mm Style 
(Polyester) 20/bag</td>
 </tr>
 <tr>
 	<td>Drosophila Culture Vials</td>
 <td>UCI</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Tricorn beaker</td>
 <td>Econo Lab Inc.</td>
 <td>B700-100</td>
 <td>Volume:100ml, 
size: 58x72mm</td>
 </tr>
 <tr>
 	<td>Yeast</td>
 <td>Red Star</td>
 <td>2751</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Nylon Mesh</td>
 <td>Genesse Scientific</td>
 <td>57-102</td>
 <td>120 micron pore size </td>
 </tr>
 <tr>
 	<td>Nylon Mesh</td>
 <td>Small Parts</td>
 <td>06-350/35</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Pipes</td>
 <td>Sigma</td>
 <td>108321-27-3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Pipes</td>
 <td>Sigma</td>
 <td>108321-27-3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Glycerol</td>
 <td>Fisher</td>
 <td>56-81-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>EGTA</td>
 <td>Sigma</td>
 <td>67-42-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>MgS04(Anhydrous)</td>
 <td>Fisher</td>
 <td>7487-88-9</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>PMSF</td>
 <td>Sigma</td>
 <td>329-98-6</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Leupeptin</td>
 <td>Sigma</td>
 <td><a href="http://www.sigmaaldrich.com/catalog/Lookup.do?N5=CAS+No.&amp;N3=mode+matchpartialmax&amp;N4=103476-89-7&amp;D7=0&amp;D10=&amp;N25=0&amp;N1=S_ID&amp;ST=RS&amp;F=PR">103476-89-7</a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Aprotonin</td>
 <td>Sigma</td>
 <td><a href="http://www.sigmaaldrich.com/catalog/Lookup.do?N5=CAS+No.&amp;N3=mode+matchpartialmax&amp;N4=9087-70-1&amp;D7=0&amp;D10=&amp;N25=0&amp;N1=S_ID&amp;ST=RS&amp;F=PR">9087-70-1</a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>TAME</td>
 <td>Sigma</td>
 <td>178403-8</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>STI</td>
 <td>Calbiochem</td>
 <td>65635</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>GTP</td>
 <td>Sigma</td>
 <td><a href="http://www.sigmaaldrich.com/catalog/Lookup.do?N5=CAS+No.&amp;N3=mode+matchpartialmax&amp;N4=36051-31-7&amp;D7=0&amp;D10=&amp;N25=0&amp;N1=S_ID&amp;ST=RS&amp;F=PR">36051-31-7</a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Taxol</td>
 <td>Sigma</td>
 <td>33069-62-4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>ATP analog 5'-adenylyl imidodiphosphate</td>
 <td>Sigma</td>
 <td>3605-31-7</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>NaCl</td>
 <td>Mallinckrodt</td>
 <td>7647-14-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>ATP</td>
 <td>Sigma</td>
 <td>74804-12-9</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Amicon Ultra Centrifugal Filters: .5 mL, 100kD cut off, 8 pack</td>
 <td>Millipore</td>
 <td>UFC510008</td>
 <td>&nbsp;</td>
 </tr>
</tbody> 
</table>

isolation and purification of kinesin from drosophila embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Watch this Scientific Journal Video about Isolation and Purification of Kinesin from Drosophila Embryos at JoVE.com

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

isolation-and-purification-of-kinesin-from-drosophila-embryos

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JoVE Journal

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12.1K Views.  University of California, Irvine. This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Video: Isolation and Purification of Kinesin from Drosophila Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos.

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given ...