Alignment Media Preparation
NH RDC are easily acquired and for the purpose of protein structure validation and refinement are often sufficient. The data can be collected on samples labeled only with 15N. An attempt is usually made to achieve alignment in two media as independent sets 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.
1) The first sample to be observed is an isotropic sample. Dilute one half of the sample by a factor of two as this will give a reference at the concentration of most aligned samples. This can be important in cases of weak dimers. Measure the pH as this will be useful in selecting media later.
2) Alignment of the protein sample in PEG(C12E5 /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 . The exact procedure is as follows: Mix 50ul of C12E5 (pentaethylene glycol monododecyl ether) with 200ul of buffer and 50ul of D20 by vortexing. Add approx 16ul of hexanol, in aliquots of 4ul 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. Prepare a sample by diluting the protein with peg 1:1 with vortexing. Let the sample sit for 1-2 hrs near the magnet to allow equilibration and alignment. Record the 2H splitting by running the s2pul expt with nuclei=lk. The range of the splitting should be around ~+/-20Hz. 3) For the second medium, 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. If the protein sample is less than the pI it will be positively charged and if it is more than the pI it will be negatively charged.
4) In cases where PEG fails (usually interacting too strongly with the protein), other media can also be used: phage, and poly acrylamide compressed gels Phage: Prepare a solution by adding 120 ul of protein solution (~0.5mM), 80 ul of 50mg/ml of Phage solution (availble from Asla Biotech Ltd) and 20ul of D2O Check the 2H splitting. This is typically 8-12Hz Compressed Gels can be made 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 130ul 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 30 degrees C on a plastic support wrapped with polyvinylidene chloride foil.