Alignment Media Preparation: Difference between revisions

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=== Brief Description  ===
=== Brief Description  ===


NH 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; 500ul of a 0.5-0.6mM 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 0.5-0.6mM sample is usually sufficient for this purpose.  


=== Data Acquisition to RDC Calculation  ===
=== Data Acquisition to 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>  


=== Alignment Media Preparation  ===
=== Alignment Media Preparation  ===
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==== Isotropic Sample Measurement  ====
==== Isotropic Sample Measurement  ====


The first sample to be observed is an isotropic sample. You may want to dilute the isotropic sample by one third (to 75% of its original concentration) to match the concentration of most aligned samples. In the case of a weak dimer, this may be important.<br>
The first sample to be observed is an isotropic sample. You may want to dilute the isotropic sample by one third (to 75% of its original concentration) to match the concentration of most aligned samples. In the case of a weak dimer, this may be important.<br>  


==== PEG Bicelle  ====
==== PEG Bicelle  ====


Alignment of the protein sample in PEG(C<sub>12</sub>E<sub>5</sub>/hexanol). This is used as a first alignement media because it produced primarily steric alignment (useful in dimer geometry predictions), the success rate is high, and it can be doped with other charged detergents to give a second alignment media .  
Alignment of the protein sample in PEG(C<sub>12</sub>E<sub>5</sub>/hexanol). This is used as a first alignment media because it produces primarily steric alignment (useful in dimer geometry predictions), the success rate is high, and it can be doped with other charged detergents to give a second alignment media .  


Chemical used for this preparation:  
Chemicals used for this preparation:  


:'''Sigma Aldrich 76437''', Pentaethylene glycol monododecyl ether (C<sub>12</sub>E<sub>5</sub> PEG)  
:'''Sigma Aldrich 76437''', Pentaethylene glycol monododecyl ether (C<sub>12</sub>E<sub>5</sub> PEG)  
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:'''Sigma Aldrich 436143''', Sodium dodecyl sulfate (SDS)
:'''Sigma Aldrich 436143''', Sodium dodecyl sulfate (SDS)


The preparation procedure is as follow:  
The preparation procedure is as follows:  


*Mix 50ul of C<sub>12</sub>E<sub>5</sub> (pentaethylene glycol monododecyl ether) with 200ul of buffer and 50ul of D<sub>2</sub>O by vortexing.<ref> Ruckert M and Otting G (2000), JACS, 122, 7793-7797</ref>  
*Mix 50µl of C<sub>12</sub>E<sub>5</sub> (pentaethylene glycol monododecyl ether) with 200µl of buffer and 50µl of D<sub>2</sub>O by vortexing.<ref> Ruckert M and Otting G (2000), JACS, 122, 7793-7797</ref>  
*Add approx 16ul of hexanol, in aliquots of 2ul with vortexing after each addition. The solution will go from clear to milky, then to translucent and viscous with lots of bubbles. Continue to add hexanol until the solution goes clear again. If it becomes milky/turbid again, you have gone past the nematic phase.  
*Add approximately 16µl of hexanol, in aliquots of 2µl with vortexing after each addition. The solution will go from clear to milky, then to translucent and viscous with lots of bubbles. Continue to add hexanol until the solution goes clear again. If it becomes milky/turbid again, you have gone past the nematic phase.  
*Sample Content:
*Sample Content:


::145 uL of Protein  
::145 µL of Protein  
::55 uL of 16% PEG stock solution  
::55 µL of 16% PEG stock solution  
::20 uL of D<sub>2</sub>O  
::20 µL of D<sub>2</sub>O  
:::Final PEG concentration is 4.2%.
:::Final PEG concentration is 4.2%.


*Record the <sup>2</sup>H splitting by running the s2pul expt with tn='lk' (for Varian instrument). The range of the splitting should be around ~+/-20Hz.
*Record the <sup>2</sup>H splitting by running the s2pul expt with tn='lk' (for Varian instruments). The range of the splitting should be around +/-20Hz.


*PEG can be doped with either cetyltrimethyl ammonium bromide (CTAB) for positively charged proteins or sodium octyl sulphate (SOS) for negatively charged proteins. Charging the medium to be like the protein prevents association and gives higher resolution spectra. A suitable ratio of PEG:CTAB/SOS is ~30:1.
*PEG can be doped with either cetyltrimethyl ammonium bromide (CTAB) for positively charged proteins or sodium octyl sulphate (SOS) for negatively charged proteins. Charging the medium to be like the protein prevents association and gives higher resolution spectra. A suitable ratio of PEG:CTAB/SOS is ~30:1.
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*Sample Content:
*Sample Content:


::145 uL of Protein  
::145 µL of Protein  
::55 uL of Pf1 phage  
::55 µL of Pf1 phage  
::20 uL of D<sub>2</sub>O  
::20 µL of D<sub>2</sub>O  
:::Final phage concentration is 12.5 mg/mL
:::Final phage concentration is 12.5 mg/mL


*Record the <sup>2</sup>H splitting by running the s2pul expt with tn=lk (for Varian instrument). The range of the splitting should be around ~+/-8~10 Hz.
*Record the <sup>2</sup>H splitting by running the s2pul expt with tn='lk' (for Varian instruments). The range of the splitting should be around ~+/-8~10 Hz.


==== Polyacrylamide Gel (Compressed and Stretched)  ====
==== Polyacrylamide Gel (Compressed and Stretched)  ====
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*To introduce negative charges, use acrylic acid or 2-acrylamido-2-methyl-1-propanesulfonic acid. Positive charge can be introduced by addition of (3-acrylamidopropyl)-trimethylammonium chloride (APTMAC) or N-(2-acryloamidoethyl) triethylammonium iodide. For neutral gels omit the charged species; for zwitterionic gels use equivalent amounts of acrylic acid and APTMAC. For 50% charged gels use 50% content of the charged specie and 50% acrylamide.
*To introduce negative charges, use acrylic acid or 2-acrylamido-2-methyl-1-propanesulfonic acid. Positive charge can be introduced by addition of (3-acrylamidopropyl)-trimethylammonium chloride (APTMAC) or N-(2-acryloamidoethyl) triethylammonium iodide. For neutral gels omit the charged species; for zwitterionic gels use equivalent amounts of acrylic acid and APTMAC. For 50% charged gels use 50% content of the charged specie and 50% acrylamide.


*Add 130ul of the mixture to plastic tubes with a 3.2 mm inner diameter and keep them overnight allowing polymerization to occur.
*Add 130µl of the mixture to plastic tubes with a 3.2 mm inner diameter and keep them overnight allowing polymerization to occur.


*Wash the polymerized gels extensively in deionized water (three cycles over a period of 2 days). The gels will increase in size due to electro osmotic swelling.
*Wash the polymerized gels extensively in deionized water (three cycles over a period of 2 days). The gels will increase in size due to electro osmotic swelling.

Revision as of 01:43, 27 June 2013

Brief Description

1H-15N 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 15N. Obtaining RDCs in two different alignment media greatly improve the quality of refinement and can aid in dimer structure determination; 500µl of a 0.5-0.6mM sample is usually sufficient for this purpose.

Data Acquisition to RDC Calculation

Refer to the linked pages for detailed descriptions of the Jmodulation Experiment RDC and HSQCTROSY RDC Measurement and RDC calculation methods.

Alignment Media Preparation

Isotropic Sample Measurement

The first sample to be observed is an isotropic sample. You may want to dilute the isotropic sample by one third (to 75% of its original concentration) to match the concentration of most aligned samples. In the case of a weak dimer, this may be important.

PEG Bicelle

Alignment of the protein sample in PEG(C12E5/hexanol). This is used as a first alignment media because it produces primarily steric alignment (useful in dimer geometry predictions), the success rate is high, and it can be doped with other charged detergents to give a second alignment media .

Chemicals used for this preparation:

Sigma Aldrich 76437, Pentaethylene glycol monododecyl ether (C12E5 PEG)
Sigma Aldrich H13303, Hexanol
Sigma Aldrich 855820, Cetyltrimethylammonium bromide (CTAB)
Sigma Aldrich O4003, Sodium octyl sulfate (SOS)
Sigma Aldrich 436143, Sodium dodecyl sulfate (SDS)

The preparation procedure is as follows:

  • Mix 50µl of C12E5 (pentaethylene glycol monododecyl ether) with 200µl of buffer and 50µl of D2O by vortexing.[1]
  • Add approximately 16µl of hexanol, in aliquots of 2µl with vortexing after each addition. The solution will go from clear to milky, then to translucent and viscous with lots of bubbles. Continue to add hexanol until the solution goes clear again. If it becomes milky/turbid again, you have gone past the nematic phase.
  • Sample Content:
145 µL of Protein
55 µL of 16% PEG stock solution
20 µL of D2O
Final PEG concentration is 4.2%.
  • Record the 2H splitting by running the s2pul expt with tn='lk' (for Varian instruments). The range of the splitting should be around +/-20Hz.
  • PEG can be doped with either cetyltrimethyl ammonium bromide (CTAB) for positively charged proteins or sodium octyl sulphate (SOS) for negatively charged proteins. Charging the medium to be like the protein prevents association and gives higher resolution spectra. A suitable ratio of PEG:CTAB/SOS is ~30:1.

Pf1 Phage

Preparation of a Pf1 phage alignment sample is fairly straightforward.[2] The protein sample is diluted by the alignment medium.

Chemical used for this preparation:

ASLA Biotech P-50-P, Pf1 phage 50 mg/mL

The preparation procedure is as follows:

  • Start with a protein stock 1-2 mM and a pf1 phage stock of 50 mg/mL. Prepare a sample of 12.5 mg/mL of phage.
  • Sample Content:
145 µL of Protein
55 µL of Pf1 phage
20 µL of D2O
Final phage concentration is 12.5 mg/mL
  • Record the 2H splitting by running the s2pul expt with tn='lk' (for Varian instruments). The range of the splitting should be around ~+/-8~10 Hz.

Polyacrylamide Gel (Compressed and Stretched)

Chemicals used in preparing this alignment medium can are:

Bio-Rad 161-0144, 40%Acrylamide/Bis solution 19:1
Bio-Rad 161-0733, 10X TBE
Bio-Rad 161-0700, APS
Bio-Rad 161-0800, TEMED
Sigma Aldrich M7279-25G, N,N'-methylenebisacrylamide (BIS)
Sigma Aldrich 448281, (3-Acrylamidopropyl)trimethylammonium chloride solution
Sigma Aldrich 282731, 2-Acrylamido-2-methyl-1-propanesulfonic acid
Compressed Gels - Positive, Negative and Neutral
  • Mix stock solutions of 40% acrylamide and N,N'-methylenebisacrylamide in a 19:1 ratio with 40% charged acrylate derivatives containing N,N'-methylenebisacrylamide as desired in a 19:1 ratio.
  • Dilute the mixtures 10x with TBE buffer (0.9 M TRIS, 0.9 M borate, 0.02 M EDTA, pH 8.2) to a final 7% concentration.
  • Polymerization is initiated by the addition of 0.15% ammoniumperoxide sulfate and 1% tetramethylethylenediamine (TEMED).
  • To introduce negative charges, use acrylic acid or 2-acrylamido-2-methyl-1-propanesulfonic acid. Positive charge can be introduced by addition of (3-acrylamidopropyl)-trimethylammonium chloride (APTMAC) or N-(2-acryloamidoethyl) triethylammonium iodide. For neutral gels omit the charged species; for zwitterionic gels use equivalent amounts of acrylic acid and APTMAC. For 50% charged gels use 50% content of the charged specie and 50% acrylamide.
  • Add 130µl of the mixture to plastic tubes with a 3.2 mm inner diameter and keep them overnight allowing polymerization to occur.
  • Wash the polymerized gels extensively in deionized water (three cycles over a period of 2 days). The gels will increase in size due to electro osmotic swelling.
  • Dry the gels over a 2 day period at room temperature on a teflon pan.

References

  1. ↑ Ruckert M and Otting G (2000), JACS, 122, 7793-7797
  2. ↑ Hansen MR, Mueller L, Pardi A (1998), Nat Struct Biol, 5, 1065-1074




Updated by Hsiau-Wei Lee, 2011

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