User:Andy Maloney/Kinesin & Microtubule Page/Microtubule papers/Analysis of the migration behavior of single microtubules in electric fields

Disclaimer

These are my notes. Please read the paper before reading my notes.

Paper

Analysis of the migration behaviour of single microtubules in electric fields.

Notes

• Microtubules move towards anode (I think they meant the cathode) in an electric field with a mobility of ~ 2.6 x 10^-4 cm²/V s.
  • This corresponds to an unbalanced negative charge of 0.19 electrons/tubulin dimer.
    • This is a factor of 50 difference than crystallographic data for non-assembled tubulin dimers.
  • They say that when tubulin polymerizes, it causes changes in the charge distribution of the dimer.
• Tubulin was purified from porcine brain.
• Kinesin was purified from porcine brain.
• They were able to vary the field from 0 - 100 Vdc.
• Their PEM buffer is:
  • 20 mM PIPES
  • 80 mM NaCl
  • 1 mM EGTA
  • 0.5 mM MgCl₂
  • pH 6.8
  • 10 µM Taxol
  • Ionic strength = 120 mM
• The assays used:
  • 40 µg/mL tubulin
  • 70 µg/mL kinesin
  • 5 mg/mL BSA
• You can get the crystallographic data for tubulin for CHARMM and TINKER!
• Microtubules disassembled when the ionic strength of the buffer was below 3 mM.
• Microtubules have protrusions that are 12-16 amino acids long which can be as long as 4 nm.
• Isoelectric points of tubulin:
  • alpha = 5.45 - 5.65
  • beta = 5.30 - 5.45
  • So, somewhere around these pH values, microtubules should stop moving in an electric field. But, they saw that it took a pH value of 4.1 to stop them.
• Huh, when using Taxol, a hydrophilic cleft in beta tubulin turns into a hydrophilic surface.
• They did not see a preferential orientation for microtubules in an electric field.
  • Wow, this is a negative result that was published.
• They do note that when polymerizing microtubules, a pulsed electric field will align microtubules in a preferential orientation.