User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/05/25/Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters

Disclaimer

These are my notes. Please read the paper before reading my notes as I may have missed something you may find crucial to your research.

Paper

Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters

Notes

• The introduction is a spectacular mini review of kinesin.
• Typical conventional kinesin speeds are around 600-800nm/s.
• "There is still a remarkable discrepancy between motility rates measured in cell-free systems and within neurons for fast anterograde transport."
• They used porcine brain kinesin.
• They used phosphocellulose-purified tubulin from something.
• Buffer (IEM)
  • 50 mM Imidazole
  • 0.5 mM EGTA
  • 0.1 mM EDTA
  • 0.5 mM MgCl₂
  • 0.5 mM Dithiothreitol
• They used 270 µg/mL kinesin
• They used 5 mg/mL BSA as their passivation.
• No Mg²⁺ in solution caused a reversible stop of gliding speeds.
• Substoichiometric Mg²⁺/ATP ratios caused the gliding speed to slow.
• They show a maximum speed increase with a 5:1 Mg²⁺:ATP ratio.
• They are able to see motility 3 hours after sealing the flow cell with an excess of ATP.ADP, gliding speed is reduced.
• Oh yeah, they state that protein conformation and protein-protein interactions depend on
  • pH
  • Ionic Strength
  • Temperature
• There was a slight speed maximum at a pH of 7.2 to 7.4. Why the hell are we using PIPES then?
• They used NaCl for their ionic strength change agent and saw no change in speeds.
• Temperature on the other hand...
  • There was a speed increase going from 22˚C to 37˚C but at 40˚C, the experiment failed.
  • They do not show a plot with temperature dependence.
• They do talk about kinesin density and how that affects gliding speeds.
  • They make lots of assumptions about the coverage of kinesin on their slides.
  • They do show that the more kinesin you have on a slide, the slower a microtubule will glide at. 20 µg/mL ~ 800 nm/s while 50 µg/mL ~ 700 nm/s.