Alignment Media Preparation

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.
• 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. 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

The preparation procedure is as follows:

• Prepare the positively and negatively charged 40% 19:1 bis:acrylamide solutions.
• Positively charged stock solution (2mL):

31 mg of N,N'-methylenebisacrylamide (BIS)

961 µL of (3-acrylamidopropyl)trimethylammonium chloride solution

1039 µL of H₂O

• Negatively charged stock solution (2mL):

31 mg of N,N'-methylenebisacrylamide (BIS)

802 mg of 2-acrylamido-2-methyl-1-propanesulfonic acid

2000 µL of H₂O

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

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

• 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% or 5% concentration for compressed or stretched gels, respectively.

• Polymerization is initiated by the addition of ammoniumperoxide sulfate (APS) (0.15% or 0.1% for compressed or stretched, respectively) and tetramethylethylenediamine (TEMED) (0.1% or 0.05% for compressed or stretched, respectively).
  

• Pipet the mixture into the casting tubes carefully, to avoid bubbles, and keep them 1-2 hrs, allowing polymerization to occur.
• Use a 200 µL pipet with a trimmed pipet tip (to avoid hitting the gel) and water to carefully push the compressed gels out of the casting tubes into a 1L Erlenmeyer flask filled with deionized water. For the stretched gels, use a 1000 µL pipet to carefully push the stretched gels out of the casting tubes into prepared racks (no more than 4-5 gels per rack) braced in 2L nalgene beakers filled with deionized water.
  

• 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. Use cheesecloth to drain the water from the flask for the compressed gels. Be careful when draining and adding new deionized water not to damage the gels. The racks holding the stretched gels may be moved to a new 2L nalgene beaker with fresh deionized water.
  
• 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). The buffer should not contain only the major buffering species and no salts.
  
• 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

Updated by Hsiau-Wei Lee, 2011
Updated by Kari Pederson 2013 (in progress)