HighPoint/CannonLab:Lipid Vesicle Preparation: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(New page: {{CannonLabMenu}} ==Lipid Film Preparation== ===Materials=== * CHCl<sub>3</sub> * MeOH * dry lipids * screw top glass vial with teflon seal ===Procedure=== # Make 8mL of 3:1 CHCl3:MeO...) |
(No difference)
|
Revision as of 09:50, 16 February 2012
<owwmenu font="arial, helvetica, sans-serif" bold="1"
color="white" bgcolor="indigo" hovercolor="white" bghovercolor="gray" topfontsize="10" fontSize="8" pagewidth="750" image="CannonLabIMG01.JPG" lab="MGSC/CannonLab"> Home=Lab-Home Lab Members=#, People=People, Contact=Contact Information, Associates=Collaborators and Former Members Research=#,Protein Membrane=Protein Membrane Interactions, Osmolytes=Osmolyte Effects Protocols=#, Vesicles=Lipid Vesicle Preparation, Solutions=Buffer and Osmolyte Solutions, Fluorescence=Fluorescence Spectrometry, UV-Vis=UV-Vis Spectrometry, Pipetting=Pipetting Exercise Notebooks=#, Master List=Notebook, Dr Cannon=Notebook/Jonathan Cannon Links=#, Courses=Course Pages
</owwmenu>
Lipid Film Preparation
Materials
- CHCl3
- MeOH
- dry lipids
- screw top glass vial with teflon seal
Procedure
- Make 8mL of 3:1 CHCl3:MeOH (6mL CHCl3/2mL MeOH)
- Add 2.5mL of CHCl3:MeOH solution to 25 mg lipids
- Take 1mL (10 mg lipid) of lipid/CHCl3:MeOH solution and place in 4mL vial (2 dram?)
- Dry using rotovap
- Attach vial to rotovap with a rubber septum (silicone septa only work 2-3 times before shrinking)
- Insert needle through septum to allow solvent to evaporate
- Evaporation takes <<30 min if conditions are right
- Place vial in vacuum desicator overnight to remove remainder of solvent
- Close tightly and store aliquot in freezer
Lipid Film Hydration
- Prepare the buffer appropriate for your experiments
- Determine the concentration of lipids you wish to use in your experiments
- Prepare a hydrated lipid suspension much more concentrated than the desired experimental concentration
- A concentration of 10 mg/mL is often a good starting point
- add 1 mL buffer to 10 mg lipid
- tape vial to vortexer and shake for 1 h
- store hydrated lipids in freezer until you are ready to make LUVs
- A concentration of 10 mg/mL is often a good starting point