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 affi...

<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>

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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Biology

12.1K visualizações.  University of California, Irvine. Este é um protocolo para isolar cinesina comprimento ativo total de Drosophila Embriões de molécula única estudos biofísicos. Nós mostramos como coletar embriões, faça o lisado embrião, e depois polimerizar microtúbulos (MTs). Cinesina é purificado por imobilizando-a sobre o MTS, fiação para baixo os complexos cinesina MT-e, em seguida libertando o cinesina a partir do MTs via adição de ATP.