WangLab:Magnetite Mcls

From OpenWetWare

Jump to: navigation, search

Contents

Magnetite MCL Preparation

Materials

  • Chloroform
  • Bath Sonicator
  • Vortexor
  • Lipid Mix [1]
  1. N-(a-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG)
  2. dilauroylphosphatidyl-choline (DLPC)
  3. dioleoylphosphatidylethanolamine-N-[3-(2-pyridyldithio)-propionate] (PDP-DOPE)
  • Lipid marker (?)
  • 10 nm magnetite nano-particles
  • Specific RDG peptides
  • Round bottom flask

Procedure

  1. Dissolve the three lipids (in a 1:2:2 molar ratio) into chloroform in the round bottom flask. Do this in a 10-20 mg of lipid/ml organic solvent ratio.
  2. Remove the organic solvent by either evaporating with dry nitrogen (only for volume of organic solvent less than 1ml) or through rotary evaporation.
  3. To make sure that all chloroform has been removed from the lipid layer, place the flask on a vacuum pump overnight. Lipid film should coat the bottom of the flask.
  4. Suspend the magnetite nano-particles in water.
  5. Hydrate dried lipid layer on the bottom of the flask with nano-particle suspended water.
  6. Vortex the lipid/water/nano-particle solution for a while.
  7. Sonicate solution for 30 mins at 28 W in a bath sonicator to reduce to single laminar liposomes.
  8. Add 2ml of MCLs to the RGD peptides in a .55 peptide to 1 PDP-DOPE molar ratio.
  9. Gently agitate this solution for 3.5 hours in a solution of 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.0).
  10. Purify the MCLs.

Links

Personal tools