अलगाव और kinesin के शोधन से ड्रोसोफिला भ्रूण
Summary
यह एक से सक्रिय पूर्ण लंबाई kinesin अलग प्रोटोकॉल है<em> ड्रोसोफिला</em> 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.
Abstract
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 °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.
Protocol
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
ि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.
Materials
| Name of the reagent or material | Company | Catalogue number | Comments |
| Agar (Granulated) | Fisher | 1423-500 | |
| Dextrose (D-Glucose) Anhydrous | Fisher | D16-3 | |
| Petri Dishes | Becton | 35 1007 | 60x15mm Style (Polyester) 20/bag |
| Drosophila Culture Vials | UCI | ||
| Tricorn beaker | Econo Lab Inc. | B700-100 | Volume:100ml, size: 58x72mm |
| Yeast | Red Star | 2751 | |
| Nylon Mesh | Genesse Scientific | 57-102 | 120 micron pore size |
| Nylon Mesh | Small Parts | 06-350/35 | |
| Pipes | Sigma | 108321-27-3 | |
| Pipes | Sigma | 108321-27-3 | |
| Glycerol | Fisher | 56-81-5 | |
| EGTA | Sigma | 67-42-5 | |
| MgS04(Anhydrous) | Fisher | 7487-88-9 | |
| PMSF | Sigma | 329-98-6 | |
| Leupeptin | Sigma | 103476-89-7 | |
| Aprotonin | Sigma | 9087-70-1 | |
| TAME | Sigma | 178403-8 | |
| STI | Calbiochem | 65635 | |
| GTP | Sigma | 36051-31-7 | |
| Taxol | Sigma | 33069-62-4 | |
| ATP analog 5′-adenylyl imidodiphosphate | Sigma | 3605-31-7 | |
| NaCl | Mallinckrodt | 7647-14-5 | |
| ATP | Sigma | 74804-12-9 | |
| Amicon Ultra Centrifugal Filters: .5 mL, 100kD cut off, 8 pack | Millipore | UFC510008 |
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