NanoBio: Making Unilamellar Vesicles - Revision history

Heiko at 16:31, 25 November 2010

Heiko — Thu, 25 Nov 2010 16:31:43 GMT

←Older revision		Revision as of 16:31, 25 November 2010
Line 58:		Line 58:
	Last updated:		Last updated:
	Caroline Ajo-Franklin 06 August 2007.		Caroline Ajo-Franklin 06 August 2007.
		+
		+	[[Category:Lipid]]
		+	[[Category:Protocol]]

CarolineAjo-Franklin: /* Procedure */

CarolineAjo-Franklin — Tue, 28 Aug 2007 19:50:58 GMT

Procedure

←Older revision		Revision as of 19:50, 28 August 2007
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	#Clean glass vials. Immerse vials in 20-30% 7x detergent (70-80% distilled water) that has been heated until clear (~90ºC) for 5 minutes. Rinse thoroughly with double distilled water. Rinse three times with acetone, then three times with chloroform. Dry with N2(g).		#Clean glass vials. Immerse vials in 20-30% 7x detergent (70-80% distilled water) that has been heated until clear (~90ºC) for 5 minutes. Rinse thoroughly with double distilled water. Rinse three times with acetone, then three times with chloroform. Dry with N2(g).
	#Add appropriate quantities of lipids in chloroform to clean glass vials. It is recommended that you keep a set of syringes to be used only for lipids.		#Add appropriate quantities of lipids in chloroform to clean glass vials. It is recommended that you keep a set of syringes to be used only for lipids.
-	#Dry the lipids down with N2(g). Ideally, the lipids will form a thin uniform film over much of the interior glass surface.	+	#Dry the lipids down with N2(g). Ideally, the lipids will form a thin, transparent, uniform film over much of the interior glass surface.
	#Dry the lipids under vacuum for 1 hour. If this is done in a desiccator, do not use desiccant.		#Dry the lipids under vacuum for 1 hour. If this is done in a desiccator, do not use desiccant.
-	#Rehydrate the lipids in buffer or Millipore water to ~10 mg/mL. Vortex the solution to redissolve as much lipid material as possible. Allow lipids to rehydrate for 15 min before extruding.	+	#Rehydrate the lipids in buffer or Millipore water to ~10 mg/mL. Vortex the solution to redissolve as much lipid material as possible. The lipid/aqueous solution should result appear cloudy. Allow lipids to rehydrate for 15 min before extruding.
	#Clean the components of extruder. Rinse pieces with absolute ethanol, allow to dry on Kimwipes, and repeat.		#Clean the components of extruder. Rinse pieces with absolute ethanol, allow to dry on Kimwipes, and repeat.
	#Assemble the extruder with filter supports (4 total) and polycarbonate membranes (2 total). See extruder instructions for more details.		#Assemble the extruder with filter supports (4 total) and polycarbonate membranes (2 total). See extruder instructions for more details.

CarolineAjo-Franklin: /* Acknowledgements */

CarolineAjo-Franklin — Tue, 28 Aug 2007 19:48:43 GMT

Acknowledgements

←Older revision		Revision as of 19:48, 28 August 2007
Line 51:		Line 51:

	==Acknowledgements==		==Acknowledgements==
-	This procedure was developed in Steve ~~Boxer’s~~ laboratory. Key contributors were Li Kung, Jennifer Hovis, and Chiaki Treynor.	+	This procedure was developed in [http://www.stanford.edu/group/boxer/ Steve Boxer's laboratory]. Key contributors were Li Kung, Jennifer Hovis, and Chiaki Treynor.

	==References==		==References==

CarolineAjo-Franklin: /* Reagents */

CarolineAjo-Franklin — Tue, 28 Aug 2007 19:45:33 GMT

Reagents

←Older revision		Revision as of 19:45, 28 August 2007
Line 7:		Line 7:
	*Lipids (usually egg PC and 1-2 mole % dye)		*Lipids (usually egg PC and 1-2 mole % dye)
	*Wide-mouth screw top glass vials, ~ 10 mL		*Wide-mouth screw top glass vials, ~ 10 mL
-	*ICN 7x detergent	+	*ICN 7x detergent, i.e MP Biomedicals catalog # 097667093
	*Millipore water		*Millipore water
	*Acetone		*Acetone

CarolineAjo-Franklin at 19:42, 28 August 2007

CarolineAjo-Franklin — Tue, 28 Aug 2007 19:42:54 GMT

←Older revision		Revision as of 19:42, 28 August 2007
Line 1:		Line 1:
-	==PREPARATION OF SMALL UNILAMELLAR VESICLES==	+	=PREPARATION OF SMALL UNILAMELLAR VESICLES=
-	~~'''~~Abstract~~'''~~	+	==Abstract==
	This procedure details how small (~35-100 nm diameter) unilamellar vesicles can be formed by extrusion.		This procedure details how small (~35-100 nm diameter) unilamellar vesicles can be formed by extrusion.

-	~~'''~~Materials~~'''~~	+	==Materials==
-	''Reagents~~:''~~	+	===Reagents===
	*Lipids (usually egg PC and 1-2 mole % dye)		*Lipids (usually egg PC and 1-2 mole % dye)
	*Wide-mouth screw top glass vials, ~ 10 mL		*Wide-mouth screw top glass vials, ~ 10 mL
Line 15:		Line 15:
	*Kimwipes		*Kimwipes

-	''Equipment~~:''~~	+	===Equipment===
	*50 uL Hamilton syringe, to be used for lipids only		*50 uL Hamilton syringe, to be used for lipids only
	*250 uL Hamilton syringe, to be used for lipids only		*250 uL Hamilton syringe, to be used for lipids only
Line 25:		Line 25:
	*Vortex		*Vortex

-	~~'''~~Procedure~~'''~~	+	==Procedure==
-	~~''Method:''~~	+
	#Clean glass vials. Immerse vials in 20-30% 7x detergent (70-80% distilled water) that has been heated until clear (~90ºC) for 5 minutes. Rinse thoroughly with double distilled water. Rinse three times with acetone, then three times with chloroform. Dry with N2(g).		#Clean glass vials. Immerse vials in 20-30% 7x detergent (70-80% distilled water) that has been heated until clear (~90ºC) for 5 minutes. Rinse thoroughly with double distilled water. Rinse three times with acetone, then three times with chloroform. Dry with N2(g).
	#Add appropriate quantities of lipids in chloroform to clean glass vials. It is recommended that you keep a set of syringes to be used only for lipids.		#Add appropriate quantities of lipids in chloroform to clean glass vials. It is recommended that you keep a set of syringes to be used only for lipids.
Line 39:		Line 38:
	#Rinse the disassembled extruder first with water, then with ethanol. Allow to dry on Kimwipes.		#Rinse the disassembled extruder first with water, then with ethanol. Allow to dry on Kimwipes.

-	~~'''~~Critical Steps~~:'''~~	+	==Critical Steps==
	*Cleanliness of the glass vials is very important. It is crucial that there is no visible aqueous liquid in the glass vials before addition of the lipids in chloroform.		*Cleanliness of the glass vials is very important. It is crucial that there is no visible aqueous liquid in the glass vials before addition of the lipids in chloroform.
	*Having a uniform film of lipids on the interior of the glass vial makes rehydration of the lipids easier.		*Having a uniform film of lipids on the interior of the glass vial makes rehydration of the lipids easier.

-	~~'''~~Troubleshooting~~:'''~~	+	==Troubleshooting==
	*The inability to form a uniform lipid film can be an indication that the glass vial is not sufficiently clean.		*The inability to form a uniform lipid film can be an indication that the glass vial is not sufficiently clean.
	*Sometimes excessive pressure can build up in the extruder, and passing the lipid solution through the membrane requires a great deal of force. When this occurs, remove the lipid solution from the syringe, re-assemble of the extruder with fresh filter supports and polycarbonate membranes, and re-extrude the lipid solution.		*Sometimes excessive pressure can build up in the extruder, and passing the lipid solution through the membrane requires a great deal of force. When this occurs, remove the lipid solution from the syringe, re-assemble of the extruder with fresh filter supports and polycarbonate membranes, and re-extrude the lipid solution.
	*If the lipid solution does not become clear and/or it requires very, very little force to pass the lipid solution through the extruder, then it is possible that the polycarbonate membranes have developed a tear. After completing 20 extrusions, disassemble the polycarbonate and check for a tear. If one is present, re-extrude the lipid solution.		*If the lipid solution does not become clear and/or it requires very, very little force to pass the lipid solution through the extruder, then it is possible that the polycarbonate membranes have developed a tear. After completing 20 extrusions, disassemble the polycarbonate and check for a tear. If one is present, re-extrude the lipid solution.

-	~~'''~~Notes~~:'''~~	+	==Notes==
	*It is important to use lipid compositions which are in the liquid phase at the temperature which the vesicles are formed. Since egg PC is fluid to ~-10C, vesicles of egg PC can be created at room temperature and stored at 4C. Other compositions may require handling at different temperatures.		*It is important to use lipid compositions which are in the liquid phase at the temperature which the vesicles are formed. Since egg PC is fluid to ~-10C, vesicles of egg PC can be created at room temperature and stored at 4C. Other compositions may require handling at different temperatures.

-	~~'''~~Acknowledgements~~:'''~~	+	==Acknowledgements==
	This procedure was developed in Steve Boxer’s laboratory. Key contributors were Li Kung, Jennifer Hovis, and Chiaki Treynor.		This procedure was developed in Steve Boxer’s laboratory. Key contributors were Li Kung, Jennifer Hovis, and Chiaki Treynor.

-	References:	+	==References==
	Cremer & Boxer J. Phys. Chem. B 1999		Cremer & Boxer J. Phys. Chem. B 1999

	Last updated:		Last updated:
	Caroline Ajo-Franklin 06 August 2007.		Caroline Ajo-Franklin 06 August 2007.

CarolineAjo-Franklin: /* PREPARATION OF SMALL UNILAMELLAR VESICLES */

CarolineAjo-Franklin — Tue, 28 Aug 2007 19:37:49 GMT

PREPARATION OF SMALL UNILAMELLAR VESICLES

←Older revision		Revision as of 19:37, 28 August 2007
Line 1:		Line 1:
	==PREPARATION OF SMALL UNILAMELLAR VESICLES==		==PREPARATION OF SMALL UNILAMELLAR VESICLES==
-	'''Abstract'''~~/br~~	+	'''Abstract'''
	This procedure details how small (~35-100 nm diameter) unilamellar vesicles can be formed by extrusion.		This procedure details how small (~35-100 nm diameter) unilamellar vesicles can be formed by extrusion.

CarolineAjo-Franklin: New page: ==PREPARATION OF SMALL UNILAMELLAR VESICLES== '''Abstract'''/br This procedure details how small (~35-100 nm diameter) unilamellar vesicles can be formed by extrusion. '''Materials''' ''R...

CarolineAjo-Franklin — Tue, 28 Aug 2007 19:37:23 GMT

New page: ==PREPARATION OF SMALL UNILAMELLAR VESICLES== '''Abstract'''/br This procedure details how small (~35-100 nm diameter) unilamellar vesicles can be formed by extrusion. '''Materials''' ''R...

New page

==PREPARATION OF SMALL UNILAMELLAR VESICLES==
'''Abstract'''/br
This procedure details how small (~35-100 nm diameter) unilamellar vesicles can be formed by extrusion.

'''Materials'''
''Reagents:''
*Lipids (usually egg PC and 1-2 mole % dye)
*Wide-mouth screw top glass vials, ~ 10 mL
*ICN 7x detergent
*Millipore water
*Acetone
*Chloroform
*Absolute ethanol
*N2(g)
*Kimwipes

''Equipment:''
*50 uL Hamilton syringe, to be used for lipids only
*250 uL Hamilton syringe, to be used for lipids only
*Hot plate
*Extruder, e.g. mini-extruder from Avanti Polar Lipids
*Filter supports
*Polycarbonate membranes
*Clean dessicator free from desiccant
*Vortex

'''Procedure'''
''Method:''
#Clean glass vials. Immerse vials in 20-30% 7x detergent (70-80% distilled water) that has been heated until clear (~90ºC) for 5 minutes. Rinse thoroughly with double distilled water. Rinse three times with acetone, then three times with chloroform. Dry with N2(g).
#Add appropriate quantities of lipids in chloroform to clean glass vials. It is recommended that you keep a set of syringes to be used only for lipids.
#Dry the lipids down with N2(g). Ideally, the lipids will form a thin uniform film over much of the interior glass surface.
#Dry the lipids under vacuum for 1 hour. If this is done in a desiccator, do not use desiccant.
#Rehydrate the lipids in buffer or Millipore water to ~10 mg/mL. Vortex the solution to redissolve as much lipid material as possible. Allow lipids to rehydrate for 15 min before extruding.
#Clean the components of extruder. Rinse pieces with absolute ethanol, allow to dry on Kimwipes, and repeat.
#Assemble the extruder with filter supports (4 total) and polycarbonate membranes (2 total). See extruder instructions for more details.
#Rinse the assembled extruder with Millipore water three times to remove any air bubbles.
#Pass the lipid solution ~20 times through the polycarbonate membranes. You should feel slight resistance when pushing the syringes, and the lipid solution should become transparent (vs. opaque).
#Store the vesicles at 4ºC.
#Rinse the disassembled extruder first with water, then with ethanol. Allow to dry on Kimwipes.

'''Critical Steps:'''
*Cleanliness of the glass vials is very important. It is crucial that there is no visible aqueous liquid in the glass vials before addition of the lipids in chloroform.
*Having a uniform film of lipids on the interior of the glass vial makes rehydration of the lipids easier.

'''Troubleshooting:'''
*The inability to form a uniform lipid film can be an indication that the glass vial is not sufficiently clean.
*Sometimes excessive pressure can build up in the extruder, and passing the lipid solution through the membrane requires a great deal of force. When this occurs, remove the lipid solution from the syringe, re-assemble of the extruder with fresh filter supports and polycarbonate membranes, and re-extrude the lipid solution.
*If the lipid solution does not become clear and/or it requires very, very little force to pass the lipid solution through the extruder, then it is possible that the polycarbonate membranes have developed a tear. After completing 20 extrusions, disassemble the polycarbonate and check for a tear. If one is present, re-extrude the lipid solution.

'''Notes:'''
*It is important to use lipid compositions which are in the liquid phase at the temperature which the vesicles are formed. Since egg PC is fluid to ~-10C, vesicles of egg PC can be created at room temperature and stored at 4C. Other compositions may require handling at different temperatures.

'''Acknowledgements:'''
This procedure was developed in Steve Boxer’s laboratory. Key contributors were Li Kung, Jennifer Hovis, and Chiaki Treynor.

References:
Cremer & Boxer J. Phys. Chem. B 1999

Last updated:
Caroline Ajo-Franklin 06 August 2007.