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

Vesicles minnesota.jpg

Fig. 1. Image of a 20 uM diameter vesicle observed under 40x objective. The Alpha-hemolysin-eGFP molecules are localized at the lipid membrane.

Vesicle brightfield.jpg Vesicle 2hr.jpg Vesicle gfp.jpg
Fig. 2a. Brightfield image of vesicles. Fig. 2b. Fluorescence image of vesicles 2 hours after incubation. Fig. 2c. Composite image of brightfield and fluorescence images.