Understanding how the differential binding of Eg5 onto spindle microtubule causes cohesion fatigue during anti-mitotic cancer treatment

• Main Author: Kwek, Milton Sheng Yi
• Other Authors: Li Hoi Yeung
• Format: Final Year Project
• Language: English
• Published: 2014
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/60306
• Institution: Nanyang Technological University

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.