Alignment Sample Preparation - Revision history

Hlee at 20:07, 1 August 2010

2010-08-01T20:07:40Z

← Older revision		Revision as of 20:07, 1 August 2010
Line 101:		Line 101:
	Dissolve to 3.5-4.5% with protein solution.  Sample should be isotropic at 25 C and aligned from 25 -30 C.  2H splitting peaks should be symmetrical and of equal height.  There should be no residual isotropic peak.  Collect 15N-1H RDCs and determine level of alignment.  Target range is at least 20 Hz, which will roughly correspond to a 2H splitting of 10-16 Hz.		Dissolve to 3.5-4.5% with protein solution.  Sample should be isotropic at 25 C and aligned from 25 -30 C.  2H splitting peaks should be symmetrical and of equal height.  There should be no residual isotropic peak.  Collect 15N-1H RDCs and determine level of alignment.  Target range is at least 20 Hz, which will roughly correspond to a 2H splitting of 10-16 Hz.
	<div></div>		<div></div>

			Modified by Hsiau-Wei Lee 07/2010

Hlee at 20:06, 1 August 2010

2010-08-01T20:06:53Z

Hlee: Created page with ' '''Phage'''
…'

2009-12-10T15:42:40Z

Created page with ' '''Phage''' …'

New page

'''Phage'''

 

Start with a protein stock 1-2 mM and a pf1 phage stock of 50 mg/mL.  Prepare a sample of 10 mg/mL.  Prepare a sample of 10 mg/mL of phage as follows:

 

250 uL protein 175 uL protein

90 uL phage 50 uL phage

45 uL D2O 25 uL D2O

65 uL buffer

total volume 450 uL total volume 250 uL

 

Collect 15N-1H RDCs and determine level of alignment.  Target range is at least  20 Hz, which will roughly correspond to a 2H splitting of 13 Hz.  If sample is not aligned strongly enough, add phage to a concentration of 15 mg/mL and repeat.

 

 

'''Peg'''

 

Start with a protein stock of 1-2 mM that contains 10% D2O and a peg bicelle stock of ~16% 

 

50 uL C12E5 (pentaethylene glycol monododecyl ether)

16 uL Hexanol

250 uL Buffer containing 10% D2O at correct pH (200 uL buffer +50 uL D2O)

 

Mix C12E5 and buffer well with vortexing.  Add hexonal in 4 uL increments, vortexing well after each addition, Solution goes from clear to milky, turbid, then to translucent and viscous with lots of bubbles.  Continue adding hexanol until solution goes clear again.  If it becomes milky/turbid again, you have gone past the nematic phase.

 

Prepare a sample of 4-6% peg by diluting protein and peg (65% protein and 35% peg) with vortexing.  Let sample sit at room temperature overnight to allow equilibration of alignment.  2H splitting peaks should be symmetrical and of equal height.  There should be no residual isotropic peak.  Collect 15N-1H RDCs and determine level of alignment.  Target range is at least  20 Hz, which will roughly correspond to a 2H splitting of 10-16 Hz.

 

'''Peg/CTAB'''

 

Start with a protein stock of 1-2 mM that contains 10% D2O and a peg bicelle stock of 16%.

 

50 uL C12E5 (pentaethylene glycol monododecyl ether)

16 uL Hexanol

250 uL Buffer containing 10% D2O at correct pH

Mix C12E5 and buffer well with vortexing.  Add hexonal in 4 uL increments, vortexing well after each addition, Solution goes from clear to milky, turbid, then to translucent and viscous with lots of bubbles.  Continue adding hexanol until solution goes clear again.  If it becomes milky/turbid again, you have gone past the nematic phase.

 

Prepare a sample of 4-6% peg by diluting protein and peg (65% protein and 35% peg) with vortexing.  Let sample sit at room temperature overnight to allow equilibration of alignment.  2H splitting peaks should be symmetrical and of equal height.  There should be no residual isotropic peak.  Collect 15N-1H RDCs and determine level of alignment.  Target range is at least  20 Hz, which will roughly correspond to a 2H splitting of 10-16 Hz.

 

Peg can be doped with CTAB to provide a second alignment tensor.  The ratio of Peg:CTAB is 27:1.

 

Peg can also be doped with SOS to provide another alignment tensor.  The Peg-SOS is now negative charged.

 

 

'''Bicelle'''

 

Start with a protein stock of 1-2 mM that contains 10% D2O and a bicelle stock of 15%.

 

62.2 mg DMPC

12.7 mg DHPC

 

Mix each lipid separately in 300 uL buffer containing D2O.  Vortex thoroughly.  DHPC should dissolve easily.  DMPC will not dissolve but rather forms a milky white suspension.  When both samples are homogeneous, add DHPC to DMPC and vortex well.  Smal white chunks may form.  Freeze with liquid N2 or dry ice/acetone bath.  Thaw to RT, vortex.  Sonicate.  Repeat at least three times and continue process until sample is clear at RT with no chunks.  If chunks remain, place bicelles in an -80 C freezer 1-2 hours, thaw, vortex.

 

Dissolve to 3.5-4.5% with protein solution.  Sample should be isotropic at 25 C and aligned from 25 -30 C.  2H splitting peaks should be symmetrical and of equal height.  There should be no residual isotropic peak.  Collect 15N-1H RDCs and determine level of alignment.  Target range is at least 20 Hz, which will roughly correspond to a 2H splitting of 10-16 Hz.
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← Older revision		Revision as of 20:06, 1 August 2010
Line 1:		Line 1:
	~~<span class="Apple-style-span" style="font-family: Arial; font-size: 12px; line-height: normal; ">~~<span style="text-decoration: underline">'''Phage'''~~</span>~~</span>		<span style="text-decoration: underline">'''Phage'''</span>

	<br>		<br>

	Start with a protein stock 1-2 mM and a pf1 phage stock of 50 mg/mL.  Prepare a sample of 10 mg/mL.  Prepare a sample of 10 mg/mL of phage as follows:		Start with a protein stock 1-2 mM and a pf1 phage stock of 50 mg/mL.  Prepare a sample of 10 mg/mL.  Prepare a sample of 10 mg/mL of phage as follows:

	<br>		<br>

	250 uL protein<span class="Apple-tab-span" style="white-space:pre"> </span>175 uL protein		250 uL protein<span class="Apple-tab-span" style="white-space:pre"> </span>175 uL protein

	90 uL phage<span class="Apple-tab-span" style="white-space:pre"> </span>50 uL phage		90 uL phage<span class="Apple-tab-span" style="white-space:pre"> </span>50 uL phage

	45 uL D2O<span class="Apple-tab-span" style="white-space:pre"> </span>25 uL D2O		45 uL D2O<span class="Apple-tab-span" style="white-space:pre"> </span>25 uL D2O

	65 uL buffer		65 uL buffer

	total volume 450 uL<span class="Apple-tab-span" style="white-space:pre"> </span>total volume 250 uL		total volume 450 uL<span class="Apple-tab-span" style="white-space:pre"> </span>total volume 250 uL

	<br>		<br>

	Collect <sup>15</sup>N-<sup>1</sup>H RDCs and determine level of alignment.  Target range is at least  20 Hz, which will roughly correspond to a <sup>2</sup>H splitting of 13 Hz.  If sample is not aligned strongly enough, add phage to a concentration of 15 mg/mL and repeat.		Collect <sup>15</sup>N-<sup>1</sup>H RDCs and determine level of alignment.  Target range is at least  20 Hz, which will roughly correspond to a <sup>2</sup>H splitting of 13 Hz.  If sample is not aligned strongly enough, add phage to a concentration of 15 mg/mL and repeat.

	<br>		<br>

	<br>		<br>

	<span style="text-decoration: underline">'''Peg'''</span>		<span style="text-decoration: underline">'''Peg'''</span>

	<br>		<br>

	Start with a protein stock of 1-2 mM that contains 10% D2O and a peg bicelle stock of ~16%		Start with a protein stock of 1-2 mM that contains 10% D2O and a peg bicelle stock of ~16%

	<br>		<br>

	50 uL C12E5 (pentaethylene glycol monododecyl ether)		50 uL C12E5 (pentaethylene glycol monododecyl ether)

	16 uL Hexanol		16 uL Hexanol

	250 uL Buffer containing 10% D<sub>2</sub>O at correct pH (200 uL buffer +50 uL D2O)		250 uL Buffer containing 10% D<sub>2</sub>O at correct pH (200 uL buffer +50 uL D2O)

	<br>		<br>

	Mix C12E5 and buffer well with vortexing.  Add hexonal in 4 uL increments, vortexing well after each addition, Solution goes from clear to milky, turbid, then to translucent and viscous with lots of bubbles.  Continue adding hexanol until solution goes clear again.  If it becomes milky/turbid again, you have gone past the nematic phase.		Mix C12E5 and buffer well with vortexing.  Add hexonal in 4 uL increments, vortexing well after each addition, Solution goes from clear to milky, turbid, then to translucent and viscous with lots of bubbles.  Continue adding hexanol until solution goes clear again.  If it becomes milky/turbid again, you have gone past the nematic phase.

	<br>		<br>

	Prepare a sample of 4-6% peg by diluting protein and peg (65% protein and 35% peg) with vortexing.  Let sample sit at room temperature overnight to allow equilibration of alignment.  <sup>2</sup>H splitting peaks should be symmetrical and of equal height.  There should be no residual isotropic peak.  Collect <sup>15</sup>N-<sup>1</sup>H RDCs and determine level of alignment.  Target range is at least  20 Hz, which will roughly correspond to a <sup>2</sup>H splitting of 10-16 Hz.		Prepare a sample of 4-6% peg by diluting protein and peg (65% protein and 35% peg) with vortexing.  Let sample sit at room temperature overnight to allow equilibration of alignment.  <sup>2</sup>H splitting peaks should be symmetrical and of equal height.  There should be no residual isotropic peak.  Collect <sup>15</sup>N-<sup>1</sup>H RDCs and determine level of alignment.  Target range is at least  20 Hz, which will roughly correspond to a <sup>2</sup>H splitting of 10-16 Hz.

	<br>		<br>

	<span style="text-decoration: underline">'''Peg/CTAB'''</span>		<span style="text-decoration: underline">'''Peg/CTAB'''</span>

	<br>		<br>

	Start with a protein stock of 1-2 mM that contains 10% D2O and a peg bicelle stock of 16%.		Start with a protein stock of 1-2 mM that contains 10% D2O and a peg bicelle stock of 16%.

	<br>		<br>

	50 uL C12E5 (pentaethylene glycol monododecyl ether)		50 uL C12E5 (pentaethylene glycol monododecyl ether)

	16 uL Hexanol		16 uL Hexanol

	250 uL Buffer containing 10% D<sub>2</sub>O at correct pH		250 uL Buffer containing 10% D<sub>2</sub>O at correct pH

	Mix C12E5 and buffer well with vortexing.  Add hexonal in 4 uL increments, vortexing well after each addition, Solution goes from clear to milky, turbid, then to translucent and viscous with lots of bubbles.  Continue adding hexanol until solution goes clear again.  If it becomes milky/turbid again, you have gone past the nematic phase.		Mix C12E5 and buffer well with vortexing.  Add hexonal in 4 uL increments, vortexing well after each addition, Solution goes from clear to milky, turbid, then to translucent and viscous with lots of bubbles.  Continue adding hexanol until solution goes clear again.  If it becomes milky/turbid again, you have gone past the nematic phase.

	<br>		<br>

	Prepare a sample of 4-6% peg by diluting protein and peg (65% protein and 35% peg) with vortexing.  Let sample sit at room temperature overnight to allow equilibration of alignment.  <sup>2</sup>H splitting peaks should be symmetrical and of equal height.  There should be no residual isotropic peak.  Collect <sup>15</sup>N-<sup>1</sup>H RDCs and determine level of alignment.  Target range is at least  20 Hz, which will roughly correspond to a <sup>2</sup>H splitting of 10-16 Hz.		Prepare a sample of 4-6% peg by diluting protein and peg (65% protein and 35% peg) with vortexing.  Let sample sit at room temperature overnight to allow equilibration of alignment.  <sup>2</sup>H splitting peaks should be symmetrical and of equal height.  There should be no residual isotropic peak.  Collect <sup>15</sup>N-<sup>1</sup>H RDCs and determine level of alignment.  Target range is at least  20 Hz, which will roughly correspond to a <sup>2</sup>H splitting of 10-16 Hz.

	<br>		<br>

	Peg can be doped with CTAB to provide a second alignment tensor.  The ratio of Peg:CTAB is 27:1.		Peg can be doped with CTAB to provide a second alignment tensor.  The ratio of Peg:CTAB is 27:1.

	<br>		<br>

	Peg can also be doped with SOS to provide another alignment tensor.  The Peg-SOS is now negative charged.		Peg can also be doped with SOS to provide another alignment tensor.  The Peg-SOS is now negative charged.

	<br>		<br>

	<br>		<br>

	<span style="text-decoration: underline">'''Bicelle'''</span>		<span style="text-decoration: underline">'''Bicelle'''</span>

	<br>		<br>

	Start with a protein stock of 1-2 mM that contains 10% D2O and a bicelle stock of 15%.		Start with a protein stock of 1-2 mM that contains 10% D2O and a bicelle stock of 15% (ether bicelles hydrolysis resistant).

	<br>		<br>

	62.2 mg DMPC		62.2 mg DMPC

	12.7 mg DHPC		12.7 mg DHPC

	<br>		<br>

	Mix each lipid separately in 300 uL buffer containing D2O.  Vortex thoroughly.  DHPC should dissolve easily.  DMPC will not dissolve but rather forms a milky white suspension.  When both samples are homogeneous, add DHPC to DMPC and vortex well.  Smal white chunks may form.  Freeze with liquid N2 or dry ice/acetone bath.  Thaw to RT, vortex.  Sonicate.  Repeat at least three times and continue process until sample is clear at RT with no chunks.  If chunks remain, place bicelles in an -80 C freezer 1-2 hours, thaw, vortex.		Mix each lipid separately in 300 uL buffer containing D2O.  Vortex thoroughly.  DHPC should dissolve easily.  DMPC will not dissolve but rather forms a milky white suspension.  When both samples are homogeneous, add DHPC to DMPC and vortex well.  Smal white chunks may form.  Freeze with liquid N2 or dry ice/acetone bath.  Thaw to RT, vortex.  Sonicate.  Repeat at least three times and continue process until sample is clear at RT with no chunks.  If chunks remain, place bicelles in an -80 C freezer 1-2 hours, thaw, vortex.

	<br>		<br>

	Dissolve to 3.5-4.5% with protein solution.  Sample should be isotropic at 25 C and aligned from 25 -30 C.  2H splitting peaks should be symmetrical and of equal height.  There should be no residual isotropic peak.  Collect 15N-1H RDCs and determine level of alignment.  Target range is at least 20 Hz, which will roughly correspond to a 2H splitting of 10-16 Hz.		Dissolve to 3.5-4.5% with protein solution.  Sample should be isotropic at 25 C and aligned from 25 -30 C.  2H splitting peaks should be symmetrical and of equal height.  There should be no residual isotropic peak.  Collect 15N-1H RDCs and determine level of alignment.  Target range is at least 20 Hz, which will roughly correspond to a 2H splitting of 10-16 Hz.
	<div~~><font class="Apple-style-span" face="Arial" size="3"><span class="Apple-style-span" style="font-size: 12px; line-height: normal;">~~		<div></div>
	~~</span></font~~></div>

Alignment Sample Preparation - Revision history

Hlee at 20:07, 1 August 2010

Hlee at 20:06, 1 August 2010

Hlee: Created page with ' '''Phage''' …'

Hlee: Created page with ' '''Phage'''
…'