Understanding how the differential binding of Eg5 onto spindle microtubule causes cohesion fatigue during anti-mitotic cancer treatment
Microtubule and kinesins plays multiple important roles in different phases of mitosis. In this study, we aim to characterize the interaction of kinesin-5 to microtubule using Ligand Tracer technology. To do this, we tried to purify kinesin-5 from whole cell lysate by immuno-precipitation. GFP B-2 a...
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| Format: | Final Year Project |
| Language: | English |
| Published: |
2014
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| Online Access: | http://hdl.handle.net/10356/60306 |
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| Institution: | Nanyang Technological University |
| Language: | English |
| Summary: | Microtubule and kinesins plays multiple important roles in different phases of mitosis. In this study, we aim to characterize the interaction of kinesin-5 to microtubule using Ligand Tracer technology. To do this, we tried to purify kinesin-5 from whole cell lysate by immuno-precipitation. GFP B-2 antibody did not bind to native Eg5-EGFP. After successful immuno-precipitation of HA-Eg5 with HA Y-11 antibody, we encountered difficulties in its elution from the sepharose beads. We managed to immobilize tubulin to polylysine coated glass slides. Ligand tracer experiments were performed using cell lysate containing HA-Eg5 with HA Y-11 FITC as the fluorescent probe. Although there is an increasing signal after addition of fluorescent analyte, the results were not conclusive. |
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