Alignment Media Preparation: Difference between revisions

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*Positively charged stock solution (2mL):
*Positively charged stock solution (2mL):


::3.4 mg of N,N'-methylenebisacrylamide (BIS)  
::40 mg of N,N'-methylenebisacrylamide (BIS)  
::30 µL of (3-acrylamidopropyl)trimethylammonium chloride solution  
::913 µL of (3-acrylamidopropyl)trimethylammonium chloride solution  
::1.8 mL of H<sub>2</sub>O
::1087 µL of H<sub>2</sub>O


*Negatively charged stock solution (2mL):
*Negatively charged stock solution (2mL):


::3.4 mg of N,N'-methylenebisacrylamide (BIS)  
::40 mg of N,N'-methylenebisacrylamide (BIS)  
::1.2 mg of 2-acrylamido-2-methyl-1-propanesulfonic acid  
::760 mg of 2-acrylamido-2-methyl-1-propanesulfonic acid  
::1.8 mL of H<sub>2</sub>O
::2000 µL of H<sub>2</sub>O


*Mix stock solutions of neutral and charged 40% acrylamide/bis 19:1 to achieve the desired overall charge ratio.<br>
*Mix stock solutions of neutral and charged 40% acrylamide/bis 19:1 to achieve the desired overall charge ratio.<br>


*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.
*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.
{| width="815" cellspacing="1" cellpadding="1" border="1" summary="*Note, the APS solution should be prepared fresh and polymerization will begin as soon as TEMED is added."
|+ Standard formulas for preparing compressed and stretched polyacrylamide gels.
|-
| Gel type
| % acrylamide
| % charged
| Vol. 40% charged stock solution (µL)
| Vol. 40% neutral stock solution (µL)
| Vol. 10x TBE (µL)
| Vol. 10% APS (µL)
| Vol. TEMED (µL)
| Vol. per casting tube (µL)
| Type of casting tube
|-
| charged compressed
| 7
| 50
| 43.75
| 43.75
| 400
| 7.5
| 5
| 140
| 2.8 mm ID&nbsp;plastic
|-
| charged stretched
| 5
| 50
| 62.5
| 62.5
| 860
| 10
| 5
| 300
| 3.2 mm ID&nbsp;glass
|-
| neutral stretched
| 5
| 0
| 0
| 125
| 860
| 10
| 5
| 490
| 4.5 mm ID&nbsp;glass
|}


*Polymerization is initiated by the addition of 0.15% ammoniumperoxide sulfate and 1% tetramethylethylenediamine (TEMED).<br>
*Polymerization is initiated by the addition of 0.15% ammoniumperoxide sulfate and 1% tetramethylethylenediamine (TEMED).<br>
Line 98: Line 148:


*Wash the polymerized gels extensively in deionized water (two cycles over a period of 1 day). The gels will increase in size due to electro-osmotic swelling.  
*Wash the polymerized gels extensively in deionized water (two cycles over a period of 1 day). The gels will increase in size due to electro-osmotic swelling.  
*Equilibrate the polymerized gels to the desired pH (to match the pH of the protein stock) by washing extensively in buffered solution (two cycles over a period of 1 day).<br>
*Equilibrate the polymerized gels to the desired pH (to match the pH of the protein stock) by washing extensively in buffered solution (two cycles over a period of 1 day).<br>  
*Wash the polymerized gels in deionized water overnight to allow them to swell to full size.
*Wash the polymerized gels in deionized water overnight to allow them to swell to full size.  
*Measure the diameter of the fully swollen gels and trim each gel to a length 5.7 times its diameter.
*Measure the diameter of the fully swollen gels and trim each gel to a length 5.7 times its diameter.



Revision as of 14:16, 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 0.5-1mM 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

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) 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)

The preparation procedure is as follows:

  • Prepare the 16% PEG stock solution by first mixing 50µl of C12E5 (pentaethylene glycol monododecyl ether) with 200µl of buffer and 50µl of D2O by vortexing.[1]
  • Add approximately 16µl of hexanol to the stock solution, 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 (for 220 µL sample):
145 µL of Protein stock solution
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.

Chemicals 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 0.5-1mM and a pf1 phage stock of 50 mg/mL. Prepare a sample of 12.5 mg/mL of phage.
  • Sample Content (for 220 µL sample):
145 µL of Protein stock solution
55 µL of Pf1 phage 50 mg/mL stock solution
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 for this preparation:

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
Positive and Negative (Compressed and Stretched) and Neutral (Stretched only)

The preparation procedure is as follows:

  • Prepare the positively and negatively charged 40% 19:1 bis:acrylamide solutions.
  • Positively charged stock solution (2mL):
40 mg of N,N'-methylenebisacrylamide (BIS)
913 µL of (3-acrylamidopropyl)trimethylammonium chloride solution
1087 µL of H2O
  • Negatively charged stock solution (2mL):
40 mg of N,N'-methylenebisacrylamide (BIS)
760 mg of 2-acrylamido-2-methyl-1-propanesulfonic acid
2000 µL of H2O
  • Mix stock solutions of neutral and charged 40% acrylamide/bis 19:1 to achieve the desired overall charge 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.
Standard formulas for preparing compressed and stretched polyacrylamide gels.
Gel type % acrylamide % charged Vol. 40% charged stock solution (µL) Vol. 40% neutral stock solution (µL) Vol. 10x TBE (µL) Vol. 10% APS (µL) Vol. TEMED (µL) Vol. per casting tube (µL) Type of casting tube
charged compressed 7 50 43.75 43.75 400 7.5 5 140 2.8 mm ID plastic
charged stretched 5 50 62.5 62.5 860 10 5 300 3.2 mm ID glass
neutral stretched 5 0 0 125 860 10 5 490 4.5 mm ID glass


  • Polymerization is initiated by the addition of 0.15% ammoniumperoxide sulfate and 1% tetramethylethylenediamine (TEMED).
  • Add 130µl of the mixture to plastic tubes with a 3.2 mm inner diameter and keep them 1-2 hrs allowing polymerization to occur.
  • Wash the polymerized gels extensively in deionized water (two cycles over a period of 1 day). The gels will increase in size due to electro-osmotic swelling.
  • Equilibrate the polymerized gels to the desired pH (to match the pH of the protein stock) by washing extensively in buffered solution (two cycles over a period of 1 day).
  • Wash the polymerized gels in deionized water overnight to allow them to swell to full size.
  • 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.

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
Updated by Kari Pederson 2013 (in progress)