Preparing the Protein

The end point of many protein purifications is SDS gel electrophoresis. The protein to be sequenced thus occurs as a band in the gel. Hunkapiller et al. (1983) gently stain the band (e.g., with Na-acetate), cut out the gel piece, and elude the protein in a dialysis chamber. The eluate is applied to a filter and then sequenced. The method is reliable but complicated and has a low yield. Prussak et al. (1989) do without a dialysis chamber and elude the protein via passive diffusion in the presence of 0.01% SDS. 

Many experimenters blot the sought-after protein from the SDS gel onto a membrane that can be put directly into the sequencing machine after the blot buffer s glycine is removed. From the lEF gel, you can also blot (for example) with protein mixtures whose components do not differ in size. Nevertheless, before blotting the lEF gel the ampholines must be washed out with perchloric acid (Hsieh et al. 1988). Some protein gets lost, and sensitive bindings (e.g., Asp-Pro) are partially hydrolyzed due to the acidic pH. 

The protein transfer from the gel to the blot should be as efficient as possible in order for the protein on the blot to be easily identifiable. The blot membrane should be stable against the reagents used for sequencing. Nitrocellulose and nylon membranes do not withstand the solvents of the Edman chemistry. The remaining choice for the experimenter is between coated glass fiber and PVDF membranes. 

(1988). Electroblotting onto Glass-fiber Filter from an Analytical Isoelectrofocusing Gel A Preparative Method for Isolating Proteins for N-terminal Sequencing, Ancd. Biochem. 170 1-8. 

Hunkapiller, M., et al. (1983). Isolation of Microgramm Quantities of Proteins from Polyacrylamide Gels for Amino Acid Sequence Analysis, Methods Enzymol. 91 227 236. 

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Prepare the protein to be modified in a non-amine-containing buffer at a slightly basic pH (i.e., avoid Tris or imidazole). The use of 0.1 M sodium phosphate, 0.15M NaCl, pH 7.2 works well for NHS ester reactions. The concentration of the protein in the reaction buffer may vary from pg/ml to mg/ml, but highly dilute solutions will result in less efficient modification yields. A protein concentration from 1 to 10 mg/ml works well in this reaction. 

Using sub-ambient temperatures for preparing the protein-ligand equilibrium mixtures and for centrifugation of the GPC spin column, the dissociation rate constant decreases and the off-rate diminishes, thereby expanding the kinetic window observable with GPC spin column screening to even weaker binders with Kd values >20 pM. 

As mentioned in Section 11.2, a special class of proteinaceous targeting constructs are those in which a therapeutic protein is used as the active drug substance. In such a preparation, the protein is redirected to the target tissue by the attachment of site-directing ligands such as those discussed in Section 11.3. For instance, interferon beta (IFN- 3) can be redirected to the liver by enz5matic desialylation in a procedure similar to that described earlier for fetuin (Section 11.3.1). The resultant asialo-IFN- 3 was found to have an in vivo anti-viral effect when tested in a hepatitis B model in athymic nude mice [54]. 

To prepare the protein for a docking procedure, the following considerations are usually taken into account ... 

Prepare the protein or macromolecule to be thiolated in a non-amine-containing buffer at pH 8.0. For the modification of ribosomal proteins (often cited in the literature) use 50 mM triethanolamine hydrochloride, 1 mM MgCl2, 50 mM KC1, pH 8. The magnesium and potassium salts are for stabilization of some ribosomal proteins. If other proteins are to be thiolated, the same buffer may be used without added salts for stabilization. Alternatively, 50 mM sodium phosphate, 0.15 M NaCl, pH 8, or 0.1 M sodium borate, pH 8.0 may be used. For the modification of polysaccharides, use 20 mM sodium borax, pH 10, to produce reactivity toward carbohydrate hydroxyl residues. Dissolve the protein to be modified at a concentration of 10 mg/ml in the reaction buffer of choice. Lower concentrations also may be used with a proportional scaling back of added 2-iminothiolane. 

We gratefully acknowledge the following contributors for their expertise in preparing the protein samples referred to in this manuscript ... 

Prepare the protein-RNA complex as described in Section 4.1 using radioactively labelled RNA. Clear the solution by centrifugation for lmin at 13,000 g, and keep the precipitate for scintillation counting. 

Sample Preparation. The protein and ligand solutions must be closely matched to prevent large heats of dilution. Although these can be removed in the data analysis, they are best kept to a minimum. If possible, the protein and ligand should be dialyzed against the same buffer solution. The ligand can be dissolved in the buffer that the protein was last dialyzed against if it is too small to dialyze. 

We thank Verena Eggli for her help in preparing the proteins and Doris Suter (Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland) for the NMR measurements. Financial support from the Swiss National Science Foundation (Swiss National Science Foundation, National Research Program NRP-47 Supramolecular Functional Materials , Project No. 4047-057548/1) is gratefully acknowledged. 

Unlike proteins where use is generally made of H, and NMR data, the NMR spectroscopy of nucleotides and nucleic acids uses H, and NMR spectra. In protein NMR studies, it is usual nowadays to prepare the protein extensively labelled with both and and also sometimes with H. These techniques have not been applied so extensively in nucleic acid NMR spectroscopy. 

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