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: Difference between revisions
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{{AndyMaloneyNotebook | {{AndyMaloneyNotebook | ||
|Description= | |Description=Paper review. [http://dx.doi.org/10.1006/cbir.1999.0515 Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters] | ||
}} | }} | ||
[[Category:Kinesin and microtubules]] | [[Category:Kinesin and microtubules]] | ||
[[Category:Kinesin and microtubule papers]] | |||
[[Category:AM Temp effects on kinesin velocity]] | [[Category:AM Temp effects on kinesin velocity]] | ||
==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== | |||
[http://dx.doi.org/10.1006/cbir.1999.0515 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 MgCl<sub>2</sub> | |||
** 0.5 mM Dithiothreitol | |||
* They used 270 µg/mL kinesin | |||
* They used 5 mg/mL BSA as their passivation. | |||
* No Mg<sup>2+</sup> in solution caused a reversible stop of gliding speeds. | |||
* Substoichiometric Mg<sup>2+</sup>/ATP ratios caused the gliding speed to slow. | |||
* They show a maximum speed increase with a 5:1 Mg<sup>2+</sup>: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. |
Latest revision as of 09:46, 30 August 2010
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
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.