Tethered Membranes - Revision history

PaulDuckworth: /* Ionophores */

2013-08-23T08:54:14Z

‎Ionophores

PaulDuckworth: /* Ionophores */

2013-08-23T08:53:36Z

‎Ionophores

Oliver: /* The tethaPlate™ */

2013-07-11T06:36:01Z

‎The tethaPlate™

Oliver: Undo revision 1184 by Oliver (talk)

2013-07-10T07:47:17Z

Undo revision 1184 by Oliver (talk)

Oliver at 07:46, 10 July 2013

2013-07-10T07:46:02Z

Oliver: /* The tethaPlate™ */

2013-07-10T07:44:18Z

‎The tethaPlate™

Oliver: /* The tethaPlate™ */

2013-07-10T07:43:24Z

‎The tethaPlate™

PaulDuckworth: /* What are Tethered Membranes? */

2013-06-24T03:41:03Z

‎What are Tethered Membranes?

PaulDuckworth: /* Ionophores */

2013-04-22T06:18:37Z

‎Ionophores

PaulDuckworth at 06:58, 15 April 2013

2013-04-15T06:58:18Z

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 [[File:patchclamp1.png|400px|thumb|right|'''Figure 4.''' A classic patch clamp experiment http://en.wikipedia.org/wiki/Patch_clamp]]
-Both natural (such as [http://en.wikipedia.org/wiki/Ionomycin ionomycin] and [http://en.wikipedia.org/wiki/A23187 calicimycin]), and artificial [http://en.wikipedia.org/wiki/Ionophore ionophores] (such as [http://en.wikipedia.org/wiki/Crown_ether crown ethers], [http://en.wikipedia.org/wiki/Calixarene calixeranes], [http://en.wikipedia.org/wiki/Cryptand cryptands] and other [http://en.wikipedia.org/wiki/Synthetic_ion_channels synthetic ion channels]) can also be studied with tethered membrane systems. Ionophores are not always polypeptides and may be quite small molecules, however they must be at  least partially lipohilic to ensure they can enter the membrane. Several ionophores have found [http://wiki.edaq.com/index.php/Ionophores_in_Society applications] as antibiotics, livestock growth promoters, ion sensors.
+Both natural (such as [http://en.wikipedia.org/wiki/Ionomycin ionomycin] and [http://en.wikipedia.org/wiki/A23187 calicimycin]), and artificial [http://en.wikipedia.org/wiki/Ionophore ionophores] (such as [http://en.wikipedia.org/wiki/Crown_ether crown ethers], [http://en.wikipedia.org/wiki/Calixarene calixeranes], [http://en.wikipedia.org/wiki/Cryptand cryptands] and other [http://en.wikipedia.org/wiki/Synthetic_ion_channels synthetic ion channels]) can also be studied with tethered membrane systems. Ionophores are not always polypeptides and may be quite small molecules, however they must be at  least partially lipohilic to ensure they can enter the membrane. Several ionophores have found [http://wiki.edaq.com/index.php/Ionophores_in_Society applications] as antibiotics, livestock growth promoters, and ion sensors.
 == Classic Patch Clamping ==

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 [[File:patchclamp1.png|400px|thumb|right|'''Figure 4.''' A classic patch clamp experiment http://en.wikipedia.org/wiki/Patch_clamp]]
-Both natural (such as [http://en.wikipedia.org/wiki/Ionomycin ionomycin] and [http://en.wikipedia.org/wiki/A23187 calicimycin]), and artificial [http://en.wikipedia.org/wiki/Ionophore ionophores] (such as [http://en.wikipedia.org/wiki/Crown_ether crown ethers], [http://en.wikipedia.org/wiki/Calixarene calixeranes], [http://en.wikipedia.org/wiki/Cryptand cryptands] and other [http://en.wikipedia.org/wiki/Synthetic_ion_channels synthetic ion channels]) can also be studied with tethered membrane systems. Ionophores are not always polypeptides and may be quite small molecules, however they must be at  least partially lipohilic to ensure they can enter the membrane.
+Both natural (such as [http://en.wikipedia.org/wiki/Ionomycin ionomycin] and [http://en.wikipedia.org/wiki/A23187 calicimycin]), and artificial [http://en.wikipedia.org/wiki/Ionophore ionophores] (such as [http://en.wikipedia.org/wiki/Crown_ether crown ethers], [http://en.wikipedia.org/wiki/Calixarene calixeranes], [http://en.wikipedia.org/wiki/Cryptand cryptands] and other [http://en.wikipedia.org/wiki/Synthetic_ion_channels synthetic ion channels]) can also be studied with tethered membrane systems. Ionophores are not always polypeptides and may be quite small molecules, however they must be at  least partially lipohilic to ensure they can enter the membrane. Several ionophores have found [http://wiki.edaq.com/index.php/Ionophores_in_Society applications] as antibiotics, livestock growth promoters, ion sensors.
 == Classic Patch Clamping ==

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 The tethaPlate has proven to be an exceptionally robust housing for the tethered membrane preparations which can be stable for stable for months when stored at 4°C, while many ion channel preparations will last for several days if not weeks (or more)!
-Assembly of a tethaPlate and making a a tethered membrane is descibed in Videos 1 and 2.
+Assembly of a tethaPlate and making a tethered membrane is descibed in Videos 1 and 2.
 {|

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 == What are Tethered Membranes? ==
 [[File:membranelabelled.png|400px|thumb|right|'''Figure 1.''' Components of a tethered membrane system]]
 Tethered membranes are planar supported [http://en.wikipedia.org/wiki/Lipid_bilayer phospholipid bilayers] held above a gold electrode  by a set of hydrophilic [http://en.wikipedia.org/wiki/Polyethylene_glycol polyethylene glycol] (PEG) chains covalently bonded to the gold surface by [http://en.wikipedia.org/wiki/Category:Organic_disulfides organic disulfide] anchors, Figure 1. A lipophilic alkane [http://en.wikipedia.org/wiki/Phytane phytanyl group] is bonded to the top of the PEG chain which acts as a scaffold around which the membrane lipids spontaneously cluster, eventually forming a continuous membrane.
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 == The tethaPlate™ ==
 A [http://www.edaq.com/product_details_page.php?product_no=SDx-T10 tethaPlate™] comprises six chambers each with a gold electrode (3.0 x 0.7 mm = 2.1 mm<sup>2</sup>) that has been pre-coated with tether and spacer molecules. Addition of a suitable phospholipid mixture is all that is required to spontaneously form the membrane. A second, uncoated, gold counter electrode sits 0.15 mm above the membrane but does not physically interact with it.

@@ Line 1: / Line 1: @@
 == What are Tethered Membranes? ==
 [[File:membranelabelled.png|400px|thumb|right|'''Figure 1.''' Components of a tethered membrane system]]
 Tethered membranes are planar supported [http://en.wikipedia.org/wiki/Lipid_bilayer phospholipid bilayers] held above a gold electrode  by a set of hydrophilic [http://en.wikipedia.org/wiki/Polyethylene_glycol polyethylene glycol] (PEG) chains covalently bonded to the gold surface by [http://en.wikipedia.org/wiki/Category:Organic_disulfides organic disulfide] anchors, Figure 1. A lipophilic alkane [http://en.wikipedia.org/wiki/Phytane phytanyl group] is bonded to the top of the PEG chain which acts as a scaffold around which the membrane lipids spontaneously cluster, eventually forming a continuous membrane.
@@ Line 17: / Line 16: @@
 == The tethaPlate™ ==
 A [http://www.edaq.com/product_details_page.php?product_no=SDx-T10 tethaPlate™] comprises six chambers each with a gold electrode (3.0 x 0.7 mm = 2.1 mm<sup>2</sup>) that has been pre-coated with tether and spacer molecules. Addition of a suitable phospholipid mixture is all that is required to spontaneously form the membrane. A second, uncoated, gold counter electrode sits 0.15 mm above the membrane but does not physically interact with it.

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 Tethered membranes are exteremely robust with the standard 10% membranes having typical lifetimes of months after preparation (if stored at 4°C between experiments). They are also very resistant to the electrical pulses and can withstand potentials within the range -500 to +800 mV, which is far greater than can be applied in traditional whole cell voltage clamping experiments.
 == The tethaPlate™ ==
-{{#ev:youtube|F5NYLPcJDOs|300|right|Video 1. How to assemble a tethaPlate cartridge}}
-{{#ev:youtube|lvmbwY-ek0E|300|right|Video 2. Making a tethered membrane}}
 A [http://www.edaq.com/product_details_page.php?product_no=SDx-T10 tethaPlate™] comprises six chambers each with a gold electrode (3.0 x 0.7 mm = 2.1 mm<sup>2</sup>) that has been pre-coated with tether and spacer molecules. Addition of a suitable phospholipid mixture is all that is required to spontaneously form the membrane. A second, uncoated, gold counter electrode sits 0.15 mm above the membrane but does not physically interact with it.
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 Assembly of a tethaPlate and making a a tethered membrane is descibed in Videos 1 and 2.
 == Membrane Proteins==

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 Together the disulfide anchor, PEG chain, and [http://en.wikipedia.org/wiki/Phytane phytanyl group] is referred to as a molecular [http://en.wikipedia.org/wiki/Tether tether] because it ties the membrane to the gold surface. In practice the tethers are separated from each other on the gold substrate by similar molecules, called spacers, that lack the lipophilic [http://en.wikipedia.org/wiki/Phytane phytanyl group]. These spacers sit under the membrane and do not have any direct contact with it. The tethers and spacers completely cover the gold surface but they are not closely packed as they are separated by the by the bulk of the organodisulfide anchor groups.
-The average distance between the tethers and spacers can contain about 9 water molecules, while the height of the membrane above the gold surface is about 4 nm (but 0.5 nm is occupied by the organodisulfide anchor).
+The average distance between the tethers and spacers can contain about 9 water molecules. The height of the membrane above the gold surface is about 3-4 nm (but 0.5 nm is occupied by the organodisulfide anchor).
 Thus the space between the membrane and the gold surface is a mixture of PEG chains, disulfide anchor groups, water molecules, various cations and anions, and small water-soluble molecules, and is collectively known as the tethaPlasm™. It is analogous to the [http://en.wikipedia.org/wiki/Cytoplasm cytoplasm] of a living cell. The application of a constant bias voltage can be used to 'charge' the tehaPlasm with an excess of cations or anions, prior to tethaPatch or tehaPod experiments (see below).

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 Thus the space between the membrane and the gold surface is a mixture of PEG chains, disulfide anchor groups, water molecules, various cations and anions, and small water-soluble molecules, and is collectively known as the tethaPlasm™. It is analogous to the [http://en.wikipedia.org/wiki/Cytoplasm cytoplasm] of a living cell. The application of a constant bias voltage can be used to 'charge' the tehaPlasm with an excess of cations or anions, prior to tethaPatch or tehaPod experiments (see below).
-Tethered Membranes are Tough!
+=== Tethered Membranes are Tough! ===
 The ratio of tethers to spaces can be varied during manufacture. The standard tethaPlate™ uses a ratio of 1:10 which normally provides the best compromise between membrane stability and flexibility. Ratios as small as 1:100 have been used if greater flexibility is required, but less than this is not recommended as membrane integrity becomes unreliable. Conversely, ratios of up to 100% can be used if exceptional stability is required.