Lipid Membrane & Vesicle preperation
The Covaris platform has the ability to create extremely homogeneous membrane preps in seconds with out resulting to syringing, dounce homogenisation or mechanical blending.. Membranes have a tendency to aggregate which causes problems down stream. By pre treating with AFA it i possible to
create more homogeneous solutions that are more dispensable.
Vesicle SOP
Graph showing improvement in Z'
Z'starting value was minus 20
Graph showing reduction in assay time
Graph showing improvement in signal windo
w
AFA has also been demonstrated to simplify vesicle manufacture and improve vesicle product.
Acoustic Vesicle Preparation:
- Suspend the cell pellet in approximately 3 ml ice-cold Tris-Sucrose buff
- er per stacker (50 mM Tris-HCl, 250 mM sucrose, pH 7.4), and pipette into a glass vial compatible with Covaris.
- For the first pulse, place the vial into the Covaris instrument at 4ºC, and pulse using the following settings:
- Duty Cycle: 20%
- Cycles/burst: 500
- Intensity: 4
- Power tracking
- Time: 10 seconds
Then, pulse for a second time, using the following settings:
- Duty Cycle: 20%
- Cycles/burst: 500
- Intensity: 6
- Power tracking
- Time: 20 seconds
- Check the cells under the microscope, to ensure complete disruption of cells.
- Add ice-cold Tris-Sucrose buffer (50 mM Tris-HCl, 250 mM sucrose, pH 7.4) to achieve a final volume of 30 ml per stacker, and centrifuge at 100,000*g for 45 minutes at 4ºC using JS-24.15 rotor.
- Re-suspend the pellet in 3 ml ice-cold Tris-Sucrose buffer per stacker, and pulse 3 more times using the following settings (Note: only the time and intensity settings vary, other settings as above):
- Pulse 3: 20 seconds, at intensity 6
- Pulse 4: 20 seconds, at intensity 6
- Pulse 5: 40 seconds, at intensity 2
- Aliquot the suspension into Eppendorf tubes (200µl per tube), snap-freeze on dry ice, and store at -80ºC until use. An aliquot should be assayed by Bio-Rad, (microtitre protocol) to determine the total protein concentration in the preparation.
Buffers and Media:
- Tris-Sucrose buffer: 50 mM Tris, 250 mM Sucrose, pH to 7.4.
- Supplement Tris-Sucrose buffer with Roche complete protease inhibitor cocktail (1 tablet per 50ml) plus 1mM PMSF. After this, check the pH and readjust to 7.4.
- Selective Medium: 500 ml MEM + Earle’s + L-Glutamine, 50 ml FBS, 5 ml non-essential aminoacid solution, 2 ml Hygromycin B (0.2 mg/ml final concentration), 0.5 ml Zeocin (0.1 mg/ml final concentration).