Protein Purification Biochem 4511 Figures Essentials of Biochemistry 3rd Ed OSU Custom Edition P i i l off Bi Principles Biochemistry h i t 5th Ed Ed M Moran ett al l Lehninger Principles of Biochemistry 5th Ed Nelson Cox Fundamentals of Biochemistry 2nd Ed Voet Voet Pratt Protein Purification old ways gizzard 1 Identify an organism organ tissue that produces a protein of interest in large quantities often impossible 2 Homogenize the tissue and extract your protein together with thousands of others p steps p until the 3 Perform numerous selective separation protein is isolated to sufficient purity 1 Identify a gene of interest amplify by PCR clone to a plasmid 2 Overexpress the protein Bacterial cells yeast y insect or mammalian cell culture 3 Break open lyse the cells to extract the protein 4 Perform selective separation steps until the protein is isolated to sufficient purity only 1 step if the protein is tagged Extracct Pure p protein Protein Purification now Protein Separation Most physical properties of proteins may be utilized to separate and purify proteins Choose properties that characterize your protein of interest and separate from other proteins based on these properties Solubility salting in and salting out by Ammonium Sulfate Ionic charge ion exchange electrophoresis Polarity Hydrophobicity hydrophobic interaction chromatography Size gel filtration chromatography dialysis Binding specificity affinity purification Gel Electrophoresis Molecules with a net charge will migrate in an electric field with a velocity proportional to 1 Charge density 2 Size 3 Shape Zonal electrophoresis Constrain ions to move within a solid support such as a thin gel between glass plates to minimize diffusion Gel electrophoresis is generally used as an analytical technique rarely as a preparative technique technique PAGE Polyacrylamide Gel Electrophoresis PAGE Polyacrylamide Gel Electrophoresis Polymerized and crossli k d acrylamide linked l id Mesh density is controlled by 1 Concentration of acrylamide and 2 Degree of crosslinking PAGE Polyacrylamide Gel Electrophoresis High density gels better separation of small proteins Low density large proteins Gradient of density both b h llarge and d small Large proteins tangle in the gel and have slower migration Density of the cross linked gel determines the size range for proteins that move well through gel SDS PAGE Denaturing Gel Electrophoresis SDS PAGE SDS PolyAcrylamide Gel Electrophoresis SDS sodium dodecyl sulfate detergent with C12 hydrocarbon chain and negatively charged sulfate SDS binds to hydrophobic regions of protein side chains SDS 1 SDS denatures d t protein t i tertiary t ti structure t t b di by disrupting ti internal i t l charge and hydrophobic interactions uniform shape 2 Overwhelms protein native charge uniform negative charge Result Mobility of proteins towards the positive electrode depends only on size SDS PAGE Sample Preparation Preparing sample for SDS PAGE denaturing gel electrophoresis SDS sodium dodecyl sulfate denaturing detergent coats protein with negative charge Reducing agent mercaptoethanol dithiothreitol break any disulfide bonds Blue Bl e dye d e to visualize is ali e the sol solvent ent front Glycerol increases density of sample so it drops to the bottom of the sample well Boil for 3 minutes to ensure maximal unfolding SDS PAGE Sample Preparation Denaturation Denaturation Add SDS and heat to disrupt tertiary structure and impart a uniform negative charge All proteins now have a uniform charge mass ratio SDS Heat Folded Unfolded SDS PAGE Sample Preparation Reduction Reduction of covalent protein disulfide bonds Common reducing agents DTT 1 4 dithiothreitol BME mercaptoethanol TCEP tris 2 carboxyethyl phosphine SDS PAGE 1 SDS unfolds the protein uniform shape 2 SDS coats all of the proteins with negative charge uniform charge g density y Only the size remains to differentiate the proteins SDS PAGE Determining Molecular Weight Use standard proteins with known g MW molecular weights Measure and plot distance migrated of protein standards vs log of their MW Draw a standard curve through the linear range Measure the distance migrated of your Unknown Locate the corresponding log MW and find the MW of the Unknown protein SDS PAGE Protein visualization Molecular Weight Ladders Protein visualization Coomassie Brilliant Blue dye complexes with the unfolded proteins detects 100 500 ng Western blotting Detect a specific protein by staining with an antibody Sensitivity depends on antibody but can be 1 1 ng Coomassie Blue Stained Gel PAGE Polyacrylamide Gel Electrophoresis Denaturing gels SDS PAGE typical example Denature proteins break up complexes break down disulfide bonds then run gel Use U tto show h what h t proteins t i are presentt in i a mixture i t Native gels Run electrophoresis under conditions that maintain folded proteins complexes associations Due to difference in charge proteins might run in opposite directions or not at all C Can b be used d tto d demonstrate t t interactions i t ti proteint i protein protein nucleic acid interactions SDS PAGE vs PAGE Native PAGE of DNA and histone octamer complex DNA Protein Only DNA Nuc DNA The shift can be because of a change in size or in charge charge or both both SDS PAGE of 7 different folded histone octamers H3 H2A H2B H4 Figures Ottesen Lab Column Chromatography Column Chromatography uses interactions between a protein mixture and a solid support for separation based on a single physical property Different physical properties can be used used Elution The process of flowing the protein out of the column Types of Chromatography Stationary phase column Ion exchange Charged stationary phase Gel filtration size based Reverse phase Hydrophobic stationary phase Affinity y specific p interactions Mobile phase solvent conditions required to elute protein depends on protein and column used Size of column amount of protein to be analyzed purified l d ifi d For ion exchange Isoelectric Point pI In I solution l ti f ld d proteins folded t i look l k lik like bi big clusters l t off iionic i charges h Each protein has an isoelectric point pI which is the pH at which the protein carries no net charge Technically pI pK1 pK2 for transitions around neutral When pH pI protein will be negatively charged When pH pI protein will be positively charged The minimal solubility of a folded protein comes where pH pI for that protein because of interactions with water as solvent Isoelectric