Biomolecular Breadboards:Protocols:Vesicles
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Vesicle Preparation
- Protocol by Vincent Noireaux, University of Minnesota, School of Physics and Astronomy
Phospholipid preparation: eggPC (Avanti 840051P, MW = 770)
- Note: Special order phospholipids in small aliquots (~25 mg), as the lipids in the bottle do not last long after opening.
- 10mg + 1ml mineral oil, vortex strongly, heat at 50C for 10 minutes.
- Prepare a sub stock at 2mg/ml (200μl at 10mg/ml + 800μl mineral oil).
- Heat at 50C for 1 hour.
- Incubate at RT for 6 hours.
Reaction composition for 90μl (100% volume):
| Volume | Contents | Note |
|---|---|---|
| 30μl | extract | |
| 6.5μl | 3-PGA buffer | |
| 2.7μl | Mg-glutamate at 100mM | (3mM final here) depends on batch |
| 1.8μl | K-glutamate at 3M | (60mM final here) depends on batch |
| 22.5μl | AA at 6mM | |
| 2μl | BSA-RITC at 10mg/ml | (3.33μM final) for visualization |
| 0.9μl | pBEST-OR2-OR1-Pr-UTR1-sigma28 at 20nM | (0.2nM final) |
| 9μl | pBEST-Ptar-UTR1-AH-eGFP at 50nM | (5nM final) |
| 4.5μl | PEG at 40% | (2% final) |
| 10.1μl | water |
Feeding solution 1 for 180μl (no PEG + extract): PEG interferes with vesicle formation
| Volume | Contents | Note |
|---|---|---|
| 40μl | S30 buffer | |
| 20μl | extract | 11% final, 5.5% in final vesicle solution after mixing with feeding 2 |
| 13μl | 3-PGA buffer | |
| 5.4μl | Mg-glutamate at 100 mM | (3mM final here) depends on batch |
| 3.6μl | K-glutamate at 3M | (60mM final here) depends on batch |
| 45μl | AA at 6mM | |
| 53μl | water |
Feeding solution 2 for 135μl (with PEG at 6% + RNase A):
| Volume | Contents | Note |
|---|---|---|
| 45μl | S30 buffer | |
| 9.75μl | 3-PGC buffer | |
| 4.05μl | Mg-glutamate at 100 mM | (3mM final here) depends on batch |
| 2.7μl | K-glutamate at 3M | (60mM final here) depends on batch |
| 33.75μl | AA at 6mM | |
| 3μl | RNase A at 50X | optional |
| 20.25μl | PEG at 40% | |
| 16.5μl | water |
Optional: RNase A (MW = 13700, from Sigma) at 33mg/ml: 2.40 mM
- A final concentration of 50 nM, stock at 2.4 mM is 48000X.
- Stock at 4800X: 10μl + 90μl water/glycerol (50/50). To get stock at 50X: 1μl stock at 4800X + 95μl water
Vesicle formation:
- Place 20μl feeding 1 in a 1.5ml tube
- Add 1μl of reaction into 400μl phospholipid solution, vortex for several seconds with a table top vortex to create an emulsion
- Add 150μl emulsion on top of the 20μl feeding 1
- Wait for the emulsion/feeding 1 interface to stabilize and flatten
- Centrifuge at 11000rpm for 10~15 seconds.
- Remove carefully the mineral oil, take 10μl of feeding 1 (where the vesicles have been formed)
- Add the 10μl vesicle solution formed in feeding 1 + 10μl feeding 2, mix gently (pipet up/down with a new tip)
- Use Frameseal to make a small chamber on a coverslip
- Blot the vesicle solution inside the chamber (try to avoid touching the sticky sides of Frameseal)
- Seal the chamber with a second coverslip; observe on microscope
Sample Images from University of Minnesota
Fig. 1. Image of a 20 uM diameter vesicle observed under 40x objective. The Alpha-hemolysin-eGFP molecules are localized at the lipid membrane.
Fig. 2. Brightfield image of vesicles.
