BISC 330L Biochemistry Spring 2015 USC Lecture 8 Friday Jan 30 2014 1 Begin Chapter 3 material Biochemistry Toolbox Techniques in Protein Analysis 330 L Chapter 3 Exploring Proteins and Proteomes Biochemistry Toolbox Techniques in Protein Purification Gaining access to proteins Purifying proteins according to 1 Solubility 2 Size 3 Charge 4 Binding Affinity Centrifugation Gel electrophoresis So you want to study Protein X Decide on a protein of interest Develop assay for Protein X Enzyme act Determine total protein in mixture Specific Act Enzyme act protein Goal Increase specific activity Now we can begin the purification process Where do we get proteins from Proteins come from Therefore a protein must be separated from the other cellular components before its function and structure can be further studied CELLS How do we extract proteins from cells In order to release the cell s contents we must first break lyse the cell membrane This process is called homogenization Sonication Detergent lysis Homogenate Cell extract Force Mortar Pestle Now what do we do spin Dounce homogenizer minced tissue in cold buffer Example of cell fractionation by Differential Centrifugation Zonal centrifugation Density gradient Zonal centrifugation separates matters according to density Often provides a better resolution of separation of particles than differential Example of DIALYSIS use to remove unwanted small molecules or salt ions Various Types of Chromatography Gel Filtration Chromatography separates macromolecules by size and or shape differences Ion exchange Chromatography separates mainly by charge differences Example of Affinity Chromatography using beads with glucose attached to isolate glucose binding protein Affinity Chromatography Separates molecules according to their affinity to bind to specific ligands i e antibodies DNA Bead can be covalently linked to the ligand that the target protein will bind Results is highly specific purification of a single protein salt or excess ligand or Principles of SDS PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis SDS PAGE Denaturing Gel SDS denatures proteins causing multimeric proteins to dissociate into their individual subunits SDS coats the all polypeptide chains with a negative charge therefore forcing the polypeptide chains into extended conformation with similar charge mass ratios SDS eliminates the effect of differences in shape and so chain length mass is the sole determinant of the migration rate in SDS gel electrophoresis Extended conformations with similar charge mass ratios Gel Electrophoresis Example of electrophoresis on vertical gel agarose or polyacrylamide which can be used to separate macromolecules DNA RNA or Proteins of different size shape or charge Polymerization of acrylamide Staining of proteins with Coomassie Blue after gel electrophoresis Isoelectric Focusing in tube gel used to separate proteins having different pI values http nationaldiagnostics com article info php articles id 65 2D Gel Electrophoresis tube gel after isoelectric focusing mounted on SDS PAGE 2 D gel electrophoresis 1 Isoelectric focusing on gel with continuous pH gradient 2 Place first gel on top of the second gel 3 Each spot on the gel represents a unique peptide that can be described based on it s charge and size Each dot represents a unique protein 2D gel electrophoresis of all proteins in E coli 2D gel electrophoresis of proteins in normal and cancer tissues End of Lecture 8