MBMB 451A Section One - Fall 2005
Methods for Working with Proteins 1. Protein Isolation A. Selection of a protein source i. tissue and cell cultures (bacteria, yeast, mammalian, etc.) ii. genetically engineered - tagged proteins, overexpression B. Solubization i. osmotic lysis - hypotonic solution swelling and bursting ii. French press - high pressure & small orifice iii. sonicator iv. homogenizer -tissue grinder, v. glass beads versus mortar & pestle vi. dounce - 2. Separation methods A. Properties that are used to separate proteins: i. charge ii. hydrophobicity iii. affinity iv. solubility & stability v. molecular weight B. differential centrifugation - S-100 versus S-30 C. precipitation/solubility i. salting in versus salting out solubility of a protein close to its pI versus the effect of salt interacting with solvent and not the protein ii. examples of a. ammonium sulfate precipitation b. PEI - poly(ethyleneimine)precipitation D. chromatography - overall example table 6-4 i. ion exchange - cation vs anion table 6-2 a. strong versus weak (effect of pH) figure 6-8 b. counterion present is important c. types of gradients and their application linear (figure 6-7), nonlinear, step (figure 6-6) ii. affinity chromatography a. ligand based - glutathione and GST covalent linking of ligand to resin figure 6-13 and 6-14 b. speciality dyes - Cibracon blue and others c. immunoaffinity - epitope tags such as FLAG, V5, etc. d. DNA - general and specific DNAs e. others (example: heparin, hydroxyapatite) iii. gel exclusion or gel filtration figure 6-9 a. separation based on size b. exclusion volume c. availability of different size limit materials iv. HIC or hydrophobic interaction chromatography compare to reverse phase chromatography v. types of resin (table 6-3) cellulose, dextran, agarose, polyacrylamide, perfusion beads 3. Stabilization of protein A. Changing buffer i. dialysis (figure 6-11) ii. ultracentrifugation B. Concentrating protein helps to maintain active protein sometimes addition of a carrier protein may help addition of glycerol C. Inhibitors i. proteases ii. phosphatase inhibitors 4. Protein Analysis A. Gel electrophoresis i. Discontinous gel figure 6-21 a. polyacrylamide: ratio of bisacrylamide to acrylamide figure 6-19 b. TEMED - free radical stablizer c. ammonium persulfate d. stacking gel pH 6.8, buffer contains glycine pK2=9.78 e. running gel is pH 8.8, sample contains bromophenol blue for tracking purposes ii. SDS-PAGE a. SDS forms a micelle around the polypeptide the size and charge of the micelle is approximately proportional to the size of the polypeptide figure 6-25 b. denatures proteins c. protein stains i. Coomassie blue ii. silver stains iii. fluorescent stains - example is fluorescamine d. gradient gels B. 2-Dimensional gels a. 1st dimension is typically by isoelectric focusing (tube gel format) use amphylotes (figure 6-26) to create an immobilized pH gradient b. 2nd dimension is usually SDS-PAGE C. Immunoblotting D. Gel-shift or EMSA (electrophoretic mobility gel shift assay) assaysAdditional Information Precipitation Equipment used for breaking up cells to obain an extract Ordering of water molecules around hydrophobic residues on the surface of a protein Solubility of a globulin-type protein close to its isoelectric point (IEP) Structures of the commonly used charged substituents on anion exchangers Gel structure of agarose Structure of repeating unit of agarose Structure of Superdex Sephadex Sephacryl HR Selectivity curves for Sephacryl HR in phosphate buffer (0.05M, pH7.0) containing NaCl (0.15M) Selectivity curves of Sephadex G-types, globular proteins Sepharose with different concentrations of agarose Properties of Superose Properties of Sephadex Properties of Sephacryl HR Manual separation of sample to surface of gel filtration column Ultrafiltration of protein solutions Use of dialysis bag
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Last updated on September 1, 2005 .
