南京大学-杨荣武生物化学-英文版-第一篇-ProteinIV.ppt

Properties of Proteins and their Research Methods Some Properties of Proteins pI value and solubility Denature and Re-nature Hydrolysis Other properties Denaturation Disruption of the normal 3D structure of a protein, such that it loses biological activity. Usually caused by heat, Chemicals or changes in pH. Usually irreversible. A cooked egg cannot be “uncooked.” Enzymatic Hydrolysis N -terminal hydrolysis C-terminal hydrolysis Carboxypeptidase A cleaves all except for P, R, K; Carboxypeptidase B cleaves only R, and K; Carboxypeptidase Y cleaves all; Carboxypeptidase C cleaves all; Internal hydrolysis Trypsin - cleaves on the C-side of K or R; Chymotrypsin - C-side of F, Y, W Experimenting with proteins… Lets say you discover a new protein: protein X You might ask the following questions: How big is the protein? What is its isoeletric point? Is it present in other cells / organisms? What is its sequence? What is its structure? 1. How big is protein X? Method: electrophoresis Separates protein molecules based on their sizes Electrophoresis Procedure: Incubate protein X with SDS so that it becomes negatively charged Load onto gel Apply voltage to the gel Stain gel with a protein dye (e.g. Coomasie Blue) This particular kind of electrophoresis is also called SDS(Polyacrylamide Gel Electrophoresis) Electrophoresis Molecular Weight Markers 2. What is protein Xs isoelectric point? Method: isoelectric focusing Separates protein molecules based on their isoelectric point Remember proteins have different charges at different pH? Procedure: Make a gel with pH gradient Load protein Find the pH where the protein stops moving High If you combine SDSand isoelectric focusing, you get 2-dimensional gel electrophoresis (2D gel) 3. Is protein X found in other cell? Method: Western blotting Cells contain many different protein, how does one identify a particular protein-of-interest from a gel? Wit