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

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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.
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