SDx Protocol 1 - Revision history

PaulDuckworth: /* Reference */

2015-01-18T07:31:33Z

‎Reference

PaulDuckworth at 07:30, 18 January 2015

2015-01-18T07:30:24Z

PaulDuckworth: /* Reference */

2015-01-18T07:28:31Z

‎Reference

PaulDuckworth: /* Presentation of Results */

2015-01-18T07:27:41Z

‎Presentation of Results

PaulDuckworth at 06:38, 19 November 2013

2013-11-19T06:38:58Z

PaulDuckworth at 06:38, 19 November 2013

2013-11-19T06:38:25Z

PaulDuckworth at 06:37, 19 November 2013

2013-11-19T06:37:40Z

PaulDuckworth: /* Presentation of Results */

2013-09-02T07:03:05Z

‎Presentation of Results

PaulDuckworth: /* Solution Preparation */

2013-08-13T08:14:51Z

‎Solution Preparation

PaulDuckworth: /* Solution Preparation */

2013-08-13T08:13:33Z

‎Solution Preparation

← Older revision		Revision as of 07:31, 18 January 2015
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	=== Reference ===		=== Reference ===

−	[https://www.researchgate.net/publication/270895100_The_Assembly_and_Use_of_Tethered_Bilayer_Lipid_Membranes_%28tBLMs%29 The Assembly and Use of Tethered Bilayer Lipid Membranes (tBLMs). Charles Cranfield], Sonia Carne, Boris Martinac, Bruce Cornell, in Dylan M Owen (ed) Methods on Membrane Lipids, Methods in Molecular Biology, 1232, 45-53, Springer 2015. [http://www.dx.doi.org/10.1007/978-1-4939-1752-5_4 DOI: 10.1007/978-1-4939-1752-5_4]	+	[https://www.researchgate.net/publication/270895100_The_Assembly_and_Use_of_Tethered_Bilayer_Lipid_Membranes_%28tBLMs%29 The Assembly and Use of Tethered Bilayer Lipid Membranes (tBLMs).] Charles Cranfield, Sonia Carne, Boris Martinac, Bruce Cornell, in Dylan M Owen (ed) Methods on Membrane Lipids, Methods in Molecular Biology, 1232, 45-53, Springer 2015. [http://www.dx.doi.org/10.1007/978-1-4939-1752-5_4 DOI: 10.1007/978-1-4939-1752-5_4]

← Older revision		Revision as of 07:30, 18 January 2015
Line 54:		Line 54:
	=== Reference ===		=== Reference ===

−	The Assembly and Use of Tethered Bilayer Lipid Membranes (tBLMs). Charles Cranfield, Sonia Carne, Boris Martinac, Bruce Cornell, in Dylan M Owen (ed) Methods on Membrane Lipids, Methods in Molecular Biology, 1232, 45-53, Springer 2015. [http://www.dx.doi.org/10.1007/978-1-4939-1752-5_4 DOI: 10.1007/978-1-4939-1752-5_4]	+	[https://www.researchgate.net/publication/270895100_The_Assembly_and_Use_of_Tethered_Bilayer_Lipid_Membranes_%28tBLMs%29 The Assembly and Use of Tethered Bilayer Lipid Membranes (tBLMs). Charles Cranfield], Sonia Carne, Boris Martinac, Bruce Cornell, in Dylan M Owen (ed) Methods on Membrane Lipids, Methods in Molecular Biology, 1232, 45-53, Springer 2015. [http://www.dx.doi.org/10.1007/978-1-4939-1752-5_4 DOI: 10.1007/978-1-4939-1752-5_4]

← Older revision		Revision as of 07:28, 18 January 2015
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	=== Reference ===		=== Reference ===

−	The Assembly and Use of Tethered Bilayer Lipid Membranes (tBLMs). Charles Cranfield, Sonia Carne, Boris Martinac, Bruce Cornell, in Dylan M Owen (ed) Methods on Membrane Lipids, Methods in Molecular Biology, 1232, pp 45-53, Springer 2015. [http://www.dx.doi.org/10.1007/978-1-4939-1752-5_4 DOI: 10.1007/978-1-4939-1752-5_4]	+	The Assembly and Use of Tethered Bilayer Lipid Membranes (tBLMs). Charles Cranfield, Sonia Carne, Boris Martinac, Bruce Cornell, in Dylan M Owen (ed) Methods on Membrane Lipids, Methods in Molecular Biology, 1232, 45-53, Springer 2015. [http://www.dx.doi.org/10.1007/978-1-4939-1752-5_4 DOI: 10.1007/978-1-4939-1752-5_4]

← Older revision		Revision as of 07:27, 18 January 2015
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	It is usual to find that there is a sudden rise in conductivity over a single decade of concentration increase. If you want better resolution on your graph then repeat the experiment using a series of test solutions prepared over this smaller concentration range.		It is usual to find that there is a sudden rise in conductivity over a single decade of concentration increase. If you want better resolution on your graph then repeat the experiment using a series of test solutions prepared over this smaller concentration range.
		+
		+
		+	=== Reference ===
		+
		+	The Assembly and Use of Tethered Bilayer Lipid Membranes (tBLMs). Charles Cranfield, Sonia Carne, Boris Martinac, Bruce Cornell, in Dylan M Owen (ed) Methods on Membrane Lipids, Methods in Molecular Biology, 1232, pp 45-53, Springer 2015. [http://www.dx.doi.org/10.1007/978-1-4939-1752-5_4 DOI: 10.1007/978-1-4939-1752-5_4]

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 Make sure you are familiar with the concept of [http://wiki.edaq.com/index.php/Tethered_Membranes tethered membranes] and use of the [http://www.edaq.com/SDx-APP1 tethaPod software].
-The buffer solution used in this protocol is PBS ([http://en.wikipedia.org/wiki/Phosphate_buffered_saline phosphate buffered saline]) solution. However equivalent protocols using different buffer solutions can also be used, eg 0.1 M KCl, Ringer's solution, [https://en.wikipedia.org/wiki/Saline_(medicine) normal saline], various [http://en.wikipedia.org/wiki/Good%27s_buffers Good's buffers], [http://en.wikipedia.org/wiki/Tris-buffered_saline TBS], etc. Many of the ionophores mentioned below contain aliphatic carboxylic acid groups which have pK<sub>a</sub>'s about 4.5 − 5. These are fully ionized at pH 6.5 or greater, and use of a suitable pH buffered solution gives greater water solubility. Ionophores that are specific for dications (Mg<sup>2+</sup>, Ca<sup>2+</sup> etc) require buffer solutions that contain significant concentrations of these ions.
+The buffer solution used in this protocol is PBS ([http://en.wikipedia.org/wiki/Phosphate_buffered_saline phosphate buffered saline]) solution. However equivalent protocols using different buffer solutions can also be used, eg 0.1 M KCl, Ringer's solution, [https://en.wikipedia.org/wiki/Saline_(medicine) normal saline], various [http://en.wikipedia.org/wiki/Good%27s_buffers Good's buffers], [http://en.wikipedia.org/wiki/Tris-buffered_saline TBS], etc. Many of the ionophores mentioned below contain aliphatic carboxylic acid groups which have pK<sub>a</sub>'s about 4.5 − 5. These are fully ionized at pH 6.5 or greater, and use of a suitable pH buffered solution gives greater water solubility. Ionophores that are specific for dications (Mg<sup>2+</sup>, Ca<sup>2+</sup>, etc.) require buffer solutions that contain significant concentrations of these ions.
 === Membrane formation ===

← Older revision		Revision as of 06:37, 19 November 2013
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	The buffer solution used in this protocol is PBS ([http://en.wikipedia.org/wiki/Phosphate_buffered_saline phosphate buffered saline]) solution. However equivalent protocols using different buffer solutions can also be used, eg 0.1 M KCl, Ringer's solution, [https://en.wikipedia.org/wiki/Saline_(medicine) normal saline], various [http://en.wikipedia.org/wiki/Good%27s_buffers Good's buffers], [http://en.wikipedia.org/wiki/Tris-buffered_saline TBS], etc. Many of the ionophores mentioned below contain aliphatic carboxylic acid groups which have pK<sub>a</sub>'s about 4.5 − 5. These are fully ionized at pH 6.5 or greater, and use of a suitable pH buffered solution gives greater water solubility.		The buffer solution used in this protocol is PBS ([http://en.wikipedia.org/wiki/Phosphate_buffered_saline phosphate buffered saline]) solution. However equivalent protocols using different buffer solutions can also be used, eg 0.1 M KCl, Ringer's solution, [https://en.wikipedia.org/wiki/Saline_(medicine) normal saline], various [http://en.wikipedia.org/wiki/Good%27s_buffers Good's buffers], [http://en.wikipedia.org/wiki/Tris-buffered_saline TBS], etc. Many of the ionophores mentioned below contain aliphatic carboxylic acid groups which have pK<sub>a</sub>'s about 4.5 − 5. These are fully ionized at pH 6.5 or greater, and use of a suitable pH buffered solution gives greater water solubility.
		+
		+	Ionophores that are specific for dications (Mg<sup>2+</sup>, Ca<sup>2+</sup> etc) require buffer solutions that contain significant concentrations of these ions.

	=== Membrane formation ===		=== Membrane formation ===

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 Derive conductivity ratios by dividing the observed conductivities by the ''baseline'' conductivity value, i.e. in the absence of ionophore.
-Prepare a graph of log of conductivity ratio versus log of ionophore concentration.
+Prepare a graph of log of conductivity ratio versus ionophore concentration, or  log of concentration, as appropriate.
 It is usual to find that there is a sudden rise in conductivity over a single decade of concentration increase. If you want better resolution on your graph then repeat the experiment using a series of test solutions prepared over this smaller concentration range.

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 ===Solution Preparation===
-Ionophores are usually supplied in milligram quantities in vials or small bottles. A stock solution is usually prepared by rinsing the contents from the manufacturer's container into a larger vessel and making up a known volume of solution. Most manufacturers supply an amount of ionophore in a slightly greater quantity (by 1-10%) than indicated on the container's label. If exact concentrations are required, weigh the manufacturer's container before and after the contents are rinsed out, and use the determined mass in your calculations.
+Ionophores are usually supplied in milligram quantities in vials or small bottles. A stock solution is usually prepared by rinsing the contents from the manufacturer's container into a larger vessel and making up a known volume of solution. Most manufacturers supply an amount of ionophore in a slightly greater quantity (by 1 − 10%) than indicated on the container's label. If exact concentrations are required, weigh the manufacturer's container before and after the contents are rinsed out, and use the determined mass in your calculations.
 Make a 1 mM stock solution of the ionophore in methanol. Long term storage of this stock solution at −20°C (i.e. in a freezer) is recommended. Overnight storage, or even for several days, can be done at 2 − 4°C (ie in a refrigerator). Allow the stock solution to return to room temperature before using.

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 Make a 1 mM stock solution of the ionophore in methanol. Long term storage of this stock solution at −20°C (i.e. in a freezer) is recommended. Overnight storage, or even for several days, can be done at 2 − 4°C (ie in a refrigerator). Allow the stock solution to return to room temperature before using.
-Now prepare a 10 µM (10000 nM) solution in PBS buffer by doing a 1:100 dilution of the stock solution. Most 'lipophillic' ionophores are either sufficiently water soluble so as to be able to achieve a 10 µM concentration, or will form a supersaturated solution long enough for you to prepare the more dilute solutions. Using the method of serial dilution to prepare solutions of 1, 10, 100, and 1000 nM in PBS buffer.
+Now prepare a 10 µM (10000 nM) solution in PBS buffer by doing a 1:100 dilution of an aliquot of the stock solution. Most 'lipophillic' ionophores are either sufficiently water soluble so as to be able to achieve a 10 µM concentration, or will form a supersaturated solution long enough for you to prepare the more dilute solutions. Using the method of serial dilution to prepare solutions of 1, 10, 100, and 1000 nM in PBS buffer.
 Note that the 10 µM solution will still contain only 1% methanol, which means that the solution is mainly water. Higher methanol concentrations can disrupt the membrane, or cause incomplete partitioning of the ionophore into the lipophilic membrane. If you need to use more concentrated ionophore solutions then you will need to make a more concentrated stock solution (eg 10 or even 100 mM, but beware that the ionophore might not be soluble at this level) and dilute this down to the desired levels.