Analysis of Proteins Lecture Notes PDF

ANALYSIS OF PROTEINS Dr. Sebnem Garip Ustaoglu  PROTEIN ANALYSIS Protein Structure Protein Activity & Amount X-ray crstallography UV Spectroscopy Nuclear magnetic by knowing the substrate resonance (NMR)...

ANALYSIS OF PROTEINS Dr. Sebnem Garip Ustaoglu  PROTEIN ANALYSIS Protein Structure Protein Activity & Amount X-ray crstallography UV Spectroscopy Nuclear magnetic by knowing the substrate resonance (NMR) concentration and Spectroscopy comparing it with a Amino acid standard composition Bradford Protein Assay Coomassie Brilliant Blue dye Acidic (Protonated) to Basic (deprotonated) by binding to protein  DETERMINATION OF PROTEIN PRIMARY STRUCTURE (AMINO ACID COMPOSITION) An important goal of molecular medicine is the identification of proteins whose presence, absence, or deficiency is associated with specific physiologic states or diseases. The primary sequence of a protein provides both a molecular fingerprint for its identification and information that can be used to identify and clone the gene or genes that encode it.  PROTEIN SEQUENCING N-Terminal Analysis C-Terminal Analysis Amino terminal end of Carboxy terminal end of polypeptide chain polypeptide chain determined determined Sanger’s Method Carboxypeptidase Edman’s Method Method EDMAN’S SEQUENCING METHOD Chromatography, Electrophoresis, Spectroscopy…etc. Edman’s Reagent Reaction @mild alkaline condition  PROTEIN PURIFICATION Highly purified protein is essential for determination of its amino acid sequence, use in biological, medical, pharmaceutical processes. Cells contain thousands of different proteins with different amounts. The isolation of a specific protein in quantities is sufficient for analysis and may require multiple successive purification techniques. Classic approaches use differences of target protein from the others such as;  Salt Precipitation (Salting Out) The solubility of protein is strongly dependent on the salt concentration (ionic strength) of the medium. At very high ionic strengths, the salt withdraws the hydrate water from the proteins and thus leads to aggregation and precipitation of the molecules (salting out). Adding salts such as ammonium sulfate (NH4)2SO4 makes it possible to separate proteins from a mixture according to their degree of solubility (fractionation).  Chromatographic Techniques Chromatographic separations partition molecules between two phases, one mobile and the other stationary. For separation of amino acids, the stationary phase, or matrix, may be a sheet of filter paper (paper chromatography) or a thin layer of cellulose, silica, or alumina (thin-layer chromatography; TLC).  Column Chromatography stationary phase; a column containing small spherical beads of modified cellulose, acrylamide, or silica whose surface typically has been coated with chemical functional groups. stationary phase matrices interact with proteins based on their charge, hydrophobicity, and ligand-binding properties. A protein mixture is applied to the column and the liquid mobile phase is percolated through it.  Size Exclusion Chromatography Size exclusion chromatography employs porous beads  Ion Exchange Chromatography In ion exchange chromatography, proteins interact with the stationary phase by charge-charge interactions. Proteins with a net positive charge at a given pH adhere to beads with negatively charged functional groups such as carboxylates or sulfates (cation exchangers). Similarly, proteins with a net negative charge adhere to beads with positively charged functional Since the net charge on a protein is determined by the pH, groups, typically tertiary or sequential elution of proteins may be achieved by changing the pH of the mobile phase. quaternary amines (anion exchangers).  Affinity Chromatography Affinity chromatography exploits the high selectivity of most proteins for their ligands. Enzymes may be purified by affinity chromatography using immobilized substrates, products, coenzymes, or inhibitors. In theory, only proteins that interact with the immobilized ligand adhere. Bound proteins are then eluted either by competition with soluble ligand or, less selectively, by disrupting protein-ligand interactions using urea, guanidine hydrochloride, mildly acidic pH, or high salt concentrations. High Pressure Chromatography (HPLC)  SDS-PAGE—polyacrylamide gel electrophoresis (PAGE) in the presence of the anionic detergent sodium dodecyl sulfate (SDS) In Protein Laboratory we will carry out SDS-PAGE and Western blotting techniques THANK YOU