Name: isolation and purification of kinesin from drosophila embryos
Uploaded: 2012-04-27T12:00:00
Description: Watch this Scientific Journal Video about Isolation and Purification of Kinesin from Drosophila Embryos at JoVE.com

िईद्भस Ngai जेसन डेल रियो और उनके समर्थन और इस परियोजना में सहायता के लिए विशेष धन्यवाद. यह काम RO1 एसपीजी के लिए GM070676 अनुदान द्वारा समर्थित किया गया.

मक्खियों की खींच और खरीदा जा सकता है और प्रयोगशाला में परिलक्षित. ड्रोसोफिला संस्कृति प्राप्त शीशियों की राशि पर निर्भर करता है, एक अक्सर संस्कृति शीशियों के &#39;फ्लिप&#39;, एक बार शीशियों अधिकतम क्षमत...

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गोजातीय मस्तिष्क सबसे व्यापक रूप से इस्तेमाल शुरू 11 सामग्री के लिए पूर्ण लंबाई kinesin शुद्ध है, हालांकि murine मस्तिष्क के रूप में 12 अच्छी तरह से इस्तेमाल किया गया है. Kinesin के स्रोत के रूप में गोजातीय मस्?...

<table border="1"><tbody><tr><td> अभिकर्मक या सामग्री के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियाँ </td></tr><tr><td> अग्रवाल (दानेदार) </td><td> मछुआ </td><td> 1423-500 </td><td></td></tr><tr><td> Dextrose निर्जल (डी ग्लुकोज) </td><td> मछुआ </td><td> D16-3 </td><td></td></tr><tr><td> पेट्री डिश </td><td> Becton </td><td> 35 1007 </td><td> 60x15mm शैली 20/bag (पॉलिएस्टर) </td></tr><tr><td> ड्रोसोफिला संस्कृति शीशियों </td><td> UCI </td><td></td><td></td></tr><tr><td> तिरंगा बीकर </td><td> Econo लैब इंक </td><td> B700-100 </td><td> मात्रा: 100ml, आकार: 58x72mm </td></tr><tr><td> खमीर </td><td> लाल सितारा </td><td> 2751 </td><td></td></tr><tr><td> नायलॉन जाल </td><td> Genesse वैज्ञानिक </td><td> 57-102 </td><td> 120 मीटरICRON ध्यान में लीन होना आकार </td></tr><tr><td> नायलॉन जाल </td><td> छोटे भागों </td><td> 06-350/35 </td><td></td></tr><tr><td> पाइप्स </td><td> सिग्मा </td><td> 108321-27-3 </td><td></td></tr><tr><td> पाइप्स </td><td> सिग्मा </td><td> 108321-27-3 </td><td></td></tr><tr><td> ग्लिसरॉल </td><td> मछुआ </td><td> 56-81-5 </td><td></td></tr><tr><td> EGTA </td><td> सिग्मा </td><td> 67-42-5 </td><td></td></tr><tr><td> MgS0 4 (निर्जल) </td><td> मछुआ </td><td> 7487-88-9 </td><td></td></tr><tr><td> PMSF </td><td> सिग्मा </td><td> 329-98-6 </td><td></td></tr><tr><td> Leupeptin </td><td> सिग्मा </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></td></tr><tr><td> Aprotonin </td><td> सिग्मा </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></td></tr><tr><td> पालतू </td><td> सिग्मा </td><td> 178,403-8 </td><td></td></tr><tr><td> एसटीआई </td><td> Calbiochem </td><td> +६५६३५ </td><td></td></tr><tr><td> जीटीपी </td><td> सिग्मा </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></td></tr><tr><td> Taxol </td><td> सिग्मा </td><td> 33069-62-4 </td><td></td></tr><tr><td> एटीपी अनुरूप 5&#39;-adenylyl imidodiphosphate </td><td> सिग्मा </td><td> 3605-31-7 </td><td></td></tr><tr><td> NaCl </td><td> Mallinckrodt </td><td> 7647-14-5 </td><td></td></tr><tr><td> एटीपी </td><td> सिग्मा </td><td> 74804-12-9 </td><td></td></tr><tr><td> अल्ट्रा amicon केन्द्रापसारक फिल्टर: 0.5 एमएल 100kD, काट, 8 पैक </td><td> Millipore </td><td> UFC510008 </td><td></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

यह एक से सक्रिय पूर्ण लंबाई kinesin अलग प्रोटोकॉल है<em&gt; ड्रोसोफिला</em&gt; एकल अणु biophysical अध्ययन के लिए भ्रूण. हम बताते हैं कैसे इकट्ठा करने के लिए भ्रूण, भ्रूण lysate, और तब सूक्ष्मनलिकाएं (टन) भाजन करना. Kinesin टन पर immobilizing, नीचे kinesin मीट्रिक टन परिसरों कताई, और तब टन से एटीपी अलावा के माध्यम kinesin जारी करके शुद्ध होता है.

अलगाव और kinesin के शोधन से ड्रोसोफिला भ्रूण

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

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Isolation and Purification of Kinesin from Drosophila Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

Midgastrula स्टेज ड्रोसोफिला भ्रूण से Neuronal संस्कृति की तैयारी

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

CD4 के अलगाव + माउस लसीका Miltenyi एमएसीएस शोधन का उपयोग नोड्स से टी कोशिकाओं

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

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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

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के अलगाव और शुद्धीकरण ड्रोसोफिला परिधीय न्यूरॉन्स चुंबकीय मनका छंटनी द्वारा

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

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ड्रोसोफिला भ्रूण में vivo centrifugation

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

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के ईमानदार इमेजिंग ड्रोसोफिला भ्रूण

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

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प्राथमिक सेल संस्कृति से ड्रोसोफिला गेसट्रुला भ्रूण

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

Purification of Transcripts and Metabolites from Drosophila Heads

देखिए और चयापचयों से शुद्धिकरण<em&gt; ड्रोसोफिला</em&gt; प्रमुखों

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

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

से प्रारंभिक चरण भ्रूण के कुशल और तेजी से अलगाव<em&gt; Arabidopsis thaliana</em&gt; बीज

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

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एक में विवो</emसे प्रोटीन परिसरों पृथक> Crosslinking दृष्टिकोण ड्रोसोफिला भ्रूण

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...