Alignment Media Preparation - Revision history

Kpederson at 20:32, 18 December 2013

2013-12-18T20:32:16Z

← Older revision		Revision as of 20:32, 18 December 2013
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	Updated by Hsiau-Wei Lee, 2011		Updated by Hsiau-Wei Lee, 2011

	Updated by Kari Pederson 2013<br>		Updated by Kari Pederson, 2013<br>

Kpederson at 20:31, 18 December 2013

2013-12-18T20:31:18Z

← Older revision		Revision as of 20:31, 18 December 2013
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	=== Data Acquisition and RDC Calculation ===		=== Data Acquisition and RDC Calculation ===

	Refer to the linked pages for detailed descriptions of the [[Jmodulation Experiment RDC]] and [[HSQCTROSY RDC Measurement]] and their respective RDC calculation methods.<br>		Refer to the linked pages for detailed descriptions of the [[Jmodulation Experiment RDC]] and [[HSQCTROSY RDC Measurement]] and their respective RDC calculation methods.<br>

	=== Alignment Media Preparation ===		=== Alignment Media Preparation ===
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	<br>		<br>

	==== References ====		=== References ===

	<references />		<references />

Kpederson: /* Data Acquisition and RDC Calculation */

2013-12-18T20:04:43Z

Data Acquisition and RDC Calculation

← Older revision		Revision as of 20:04, 18 December 2013
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	=== Data Acquisition and RDC Calculation ===		=== Data Acquisition and RDC Calculation ===

	Refer to the linked pages for detailed descriptions of the [[Jmodulation Experiment RDC]] and [[HSQCTROSY RDC Measurement]] and RDC calculation methods.<br>		Refer to the linked pages for detailed descriptions of the [[Jmodulation Experiment RDC]] and [[HSQCTROSY RDC Measurement]] and their respective RDC calculation methods.<br>

	=== Alignment Media Preparation ===		=== Alignment Media Preparation ===

Kpederson: /* Preparation of Dried Acrylamide Gels */

2013-12-18T03:33:43Z

Preparation of Dried Acrylamide Gels

← Older revision		Revision as of 03:33, 18 December 2013
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	*Select the gels which have no cracks or imperfections and measure the diameter of the fully swollen gels and trim each gel to a length 5.7 times its diameter.		*Select the gels which have no cracks or imperfections and measure the diameter of the fully swollen gels and trim each gel to a length 5.7 times its diameter.

	*Dry the gels over a 2 day period at room temperature on a teflon pan.<br>		*Dry the gels over a 2 day period at room temperature on a teflon pan. If the gels do not dry well (straight, uniform width), but do not break, you can add a few drops of deionized water to slightly rehydrate them and allow them to dry again.<br>

	===== Preparation of Aligned Compressed Gel Samples<br> =====		===== Preparation of Aligned Compressed Gel Samples<br> =====

Kpederson at 20:40, 27 June 2013

2013-06-27T20:40:20Z

← Older revision		Revision as of 20:40, 27 June 2013
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	\|}		\|}

	Note: The APS solution should be prepared fresh and polymerization will begin as soon as TEMED is added.		Note: The APS solution should be prepared fresh and polymerization will begin as soon as TEMED is added.

	<br>		<br>
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	*:300 µL of Protein stock		*:300 µL of Protein stock
	*:20 µL of D<sub>2</sub>O		*:20 µL of D<sub>2</sub>O
	*Prepare an NMR tube by washing 3 times with dichlorodimethylsilane, then rinse with deionized water and dry.		*Prepare an NMR tube by washing the inside 3 times with dichlorodimethylsilane, then rinse with deionized water and dry.
	*Place a dried stretched gel near the top of the 5mm end of the NMR tube and attach a syringe with tubing to the bottom of the tube.		*Place a dried stretched gel near the top of the 5mm end of the NMR tube and attach a syringe with tubing to the bottom of the tube.
	*Carefully pipet in the protein stock and D<sub>2</sub>O allowing the solution's surface tension to hold it in place in the tube.		*Carefully pipet in the protein stock and D<sub>2</sub>O allowing the solution's surface tension to hold it in place in the tube.
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	*Once the gel is swollen, use the syringe to gently pull the gel down to the lower stem of the tube. Insert a small rubber stopper in the bottom of the tube to prevent the gel from drying. Carefully insert a shigemi plunger in the top of the tube, to the top of the gel or the bottom of the upper stem, whichever is higher.		*Once the gel is swollen, use the syringe to gently pull the gel down to the lower stem of the tube. Insert a small rubber stopper in the bottom of the tube to prevent the gel from drying. Carefully insert a shigemi plunger in the top of the tube, to the top of the gel or the bottom of the upper stem, whichever is higher.

	<br>		<br>

	==== References ====		==== References ====

Kpederson at 17:35, 27 June 2013

2013-06-27T17:35:37Z

← Older revision		Revision as of 17:35, 27 June 2013
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	*Place a dried stretched gel near the top of the 5mm end of the NMR tube and attach a syringe with tubing to the bottom of the tube.		*Place a dried stretched gel near the top of the 5mm end of the NMR tube and attach a syringe with tubing to the bottom of the tube.
	*Carefully pipet in the protein stock and D<sub>2</sub>O allowing the solution's surface tension to hold it in place in the tube.		*Carefully pipet in the protein stock and D<sub>2</sub>O allowing the solution's surface tension to hold it in place in the tube.
	*Use the syringe to adjust the position of the solution, such that the gel is centered in the column of solution. Cap the NMR tube, and let swell at room temperature 1-2 days. Capping the tube may cause the solution to shift, it's position should be readjusted with the syringe, to keep the gel centered.		*Use the syringe to adjust the position of the solution, such that the gel is centered in the column of solution. Cap the NMR tube, and let swell at room temperature 1-2 days. Capping the tube may cause the solution to shift, it's position should be readjusted with the syringe, to keep the gel centered.
			*Once the gel is swollen, use the syringe to gently pull the gel down to the lower stem of the tube. Insert a small rubber stopper in the bottom of the tube to prevent the gel from drying. Carefully insert a shigemi plunger in the top of the tube, to the top of the gel or the bottom of the upper stem, whichever is higher.

	<br>		<br>

Kpederson at 17:31, 27 June 2013

2013-06-27T17:31:32Z

← Older revision		Revision as of 17:31, 27 June 2013
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	\|}		\|}

	*Note: The APS solution should be prepared fresh and polymerization will begin as soon as TEMED is added.~~<br>~~		Note: The APS solution should be prepared fresh and polymerization will begin as soon as TEMED is added.

	<br>		<br>

Kpederson at 17:30, 27 June 2013

2013-06-27T17:30:16Z

← Older revision		Revision as of 17:30, 27 June 2013
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	:'''Bio-Rad 161-0700''', APS		:'''Bio-Rad 161-0700''', APS
	:'''Bio-Rad 161-0800''', TEMED		:'''Bio-Rad 161-0800''', TEMED
	:'''Sigma Aldrich M7279~~-25G~~''', N,N'-methylenebisacrylamide (BIS)		:'''Sigma Aldrich M7279''', N,N'-methylenebisacrylamide (BIS)
	:'''Sigma Aldrich 448281''', (3-Acrylamidopropyl)trimethylammonium chloride solution		:'''Sigma Aldrich 448281''', (3-Acrylamidopropyl)trimethylammonium chloride solution
	:'''Sigma Aldrich 282731''', 2-Acrylamido-2-methyl-1-propanesulfonic acid		:'''Sigma Aldrich 282731''', 2-Acrylamido-2-methyl-1-propanesulfonic acid
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	*Use the syringe to adjust the position of the solution, such that the gel is centered in the column of solution. Cap the NMR tube, and let swell at room temperature 1-2 days. Capping the tube may cause the solution to shift, it's position should be readjusted with the syringe, to keep the gel centered.		*Use the syringe to adjust the position of the solution, such that the gel is centered in the column of solution. Cap the NMR tube, and let swell at room temperature 1-2 days. Capping the tube may cause the solution to shift, it's position should be readjusted with the syringe, to keep the gel centered.

	<br>		<br>

	==== References ====		==== References ====

Kpederson at 17:29, 27 June 2013

2013-06-27T17:29:36Z

← Older revision		Revision as of 17:29, 27 June 2013
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	<sup>1</sup>H-<sup>15</sup>N residual dipolar couplings (RDCs) are easily acquired for the purpose of protein structure validation and refinement. The data can be collected on samples labeled only with <sup>15</sup>N. Obtaining RDCs in two different alignment media greatly improve the quality of refinement and can aid in dimer structure determination; 500µl of a 0.5-1mM sample is usually sufficient for this purpose.		<sup>1</sup>H-<sup>15</sup>N residual dipolar couplings (RDCs) are easily acquired for the purpose of protein structure validation and refinement. The data can be collected on samples labeled only with <sup>15</sup>N. Obtaining RDCs in two different alignment media greatly improve the quality of refinement and can aid in dimer structure determination; 500µl of a 0.5-1mM sample is usually sufficient for this purpose.

	=== Data Acquisition to RDC Calculation ===		=== Data Acquisition and RDC Calculation ===

	Refer to the linked pages for detailed descriptions of the [[Jmodulation Experiment RDC]] and [[HSQCTROSY RDC Measurement]] and RDC calculation methods.<br>		Refer to the linked pages for detailed descriptions of the [[Jmodulation Experiment RDC]] and [[HSQCTROSY RDC Measurement]] and RDC calculation methods.<br>

Kpederson at 17:29, 27 June 2013

2013-06-27T17:29:08Z

← Older revision		Revision as of 17:29, 27 June 2013
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	:'''Sigma Aldrich 448281''', (3-Acrylamidopropyl)trimethylammonium chloride solution		:'''Sigma Aldrich 448281''', (3-Acrylamidopropyl)trimethylammonium chloride solution
	:'''Sigma Aldrich 282731''', 2-Acrylamido-2-methyl-1-propanesulfonic acid		:'''Sigma Aldrich 282731''', 2-Acrylamido-2-methyl-1-propanesulfonic acid

			Other materials used for this preparation:

			:'''Wilmad P-4.4965M-6.5135-0-0''', 4.4965mm +/-0.0065mm ID x 6.5135mm +/-0.0065mm. Ground polished and buffed OD. 40mm +/- 0.5mm long. Both ends wet saw cut only.

	===== Preparation of Dried Acrylamide Gels =====		===== Preparation of Dried Acrylamide Gels =====
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	\| 5		\| 5
	\| 140		\| 140
	\| 2.8 mm ID plastic		\| 2.8 mm ID  plastic
	\|-		\|-
	\| charged stretched		\| charged stretched
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	\| 5		\| 5
	\| 300		\| 300
	\| 3.2 mm ID glass		\| 3.2 mm ID  glass
	\|-		\|-
	\| neutral stretched		\| neutral stretched
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	\| 5		\| 5
	\| 490		\| 490
	\| 4.5 mm ID glass		\| 4.5 mm ID  glass
	\|}		\|}

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	Other materials required for this preparation:		Other materials required for this preparation:

	:'''Wilmad 528-PP-2.~~8MM~~/~~STEM~~''', 528-PP NMR tube sealed to a 50mm long stem section (stem dimensions 2.8mm +/-0.025mm ID x wall 0.38mm +/-0.015mm). Overall length, 8 inches. Both ends open'''.'''		:'''Wilmad 528-PP-2.8mm/Stem''', 528-PP NMR tube sealed to a 50mm long stem section (stem dimensions 2.8mm +/-0.025mm ID x wall 0.38mm +/-0.015mm). Overall length, 8 inches. Both ends open'''.'''

	:'''Wilmad 528-PP-3.~~6MM~~/~~STEM~~''', 528-PP NMR tube sealed to a 50mm long stem section (stem dimensions 3.6mm +/-0.025mm ID x wall 0.38mm +/-0.015mm). Overall length, 8 inches. Both ends open.		:'''Wilmad 528-PP-3.6mm/Stem''', 528-PP NMR tube sealed to a 50mm long stem section (stem dimensions 3.6mm +/-0.025mm ID x wall 0.38mm +/-0.015mm). Overall length, 8 inches. Both ends open.

	:'''Wilmad 528-PP-3.~~9MM~~/~~STEM~~''', 528-PP NMR tube sealed to a 50mm long stem section (stem dimensions 3.9mm +/-0.025mm ID x wall 0.38mm +/-0.015mm). Overall length, 8 inches. Both ends open.		:'''Wilmad 528-PP-3.9mm/Stem''', 528-PP NMR tube sealed to a 50mm long stem section (stem dimensions 3.9mm +/-0.025mm ID x wall 0.38mm +/-0.015mm). Overall length, 8 inches. Both ends open.

	:'''Wilmad ~~5MM~~ microprobe NMR tube style''', Upper tube 528-PP section 7 inches long sealed to a 50mm long stem section (427-PP). Both ends open.		:'''Wilmad 5mm microprobe NMR tube style''', Upper tube 528-PP section 7 inches long sealed to a 50mm long stem section (427-PP). Both ends open.

	*Sample content (stretched gel):		*Sample content (stretched gel):
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	Updated by Hsiau-Wei Lee, 2011		Updated by Hsiau-Wei Lee, 2011

	Updated by Kari Pederson 2013 ~~(in progress)~~		Updated by Kari Pederson 2013<br>

	<br>