07 ProteinTools - Protein Tools Learning Objectives •...

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Unformatted text preview: Protein Tools: Learning Objectives • Understand the techniques used to purify proteins, – The chemical principles by which the technique works – The different considerations to take into account when using each • Understand the techniques used to identify and quantify proteins. • Understand the techniques used to determine protein structure and their limitations Protein Purification Requires a Strategy Purified Proteins Provide the Material for Detailed Structural and Functional Analysis Physical Characteristics Distinguishing Proteins Page 97 Column Chromatography Mobile Phase Load Buffer (Stationary Phase) resin in column that has some property that makes your protein of interest bind to that column (buffer increases its binding) elution buffer eludes the protein off the column Elution Buffer Isoelectric Points of Several Common Proteins Table 5-2 Ion-exchange Chromatography ion exchange column will have a charged resin and the protein of interest will have an opposite charge so it will bind to the column add salt to elution buffer in order to disrupt ionic interactions so can be used to elude off column change pH of elution buffer to the other side of pi to change the net charge of the protein so the protein will elude off the column Ion Exchange Chromatography • Cation Exchanger: • Anion Exchanger: – Negatively charged column binds positively charged proteins – Positively charged column binds negatively charged proteins – Load Buffer pH < pI – Load Buffer pH > pI Gel Filtration Chromatography beeds with pores in column. any protein that is smaller than the pore can go through the tunnels; path through the column is longer any protein that is larger than the pore wont go through it and will go out the column first add buffer and keep eluding until no more proteins come off Affinity Chromatography: Most common purification strategy Ligand: Substrate adduct or analog – High binding affinity to protein – Non-reactive highly specific ligand attached to beeds/resin in the column; protein has high affinity to ligand so it binds to column add ligand to elution buffer protein will go off the ligand in column and then bind to the ligand in the elution buffer allowing it to elute of the column Affinity Tags attach the protein 1(ligand pair protein) to protein 2 and ligand 1 (ligand pair protein) to column so then the protein of interest will bind to column protein • Uses known protein – ligand pair column Target Protein ligand column OR: Target Protein • Encoded in DNA or otherwise covalently attached to the protein • Elute with the ligand • Must be removed from protein, or check functionality ALWAYS ELUTE WITH LIGAND Examples of Affinity Tags His-tag elude with imadazole (unique case, but most common) His and beeds with nickel attached to them GST-tag: Favorite Column – Ligand Protein – protein Which of the following proteins will elute last in gel filtration chromatography? A. B. C. D. Proteinase Inhibitor III Cytochrome c RNA polymerase Triose Phosphate Isomerase 3.4 kD 11.6 kD 98.8 kD 53.9 kD If you want to perform an anion-exchange purification of a protein which has a pI of 7.5, what pH will you choose for this experiment? A. pH 6.0 B. pH 7.0 C. pH 7.5 D. pH 8.0 E. pH 9.0 You are attempting to purify protein X by adding a biotin tag to it. The protein Avidin binds very tightly to biotin. How could you elute protein X off the affinity column? I. Avidin in the elution buffer II. Biotin in the elution buffer III. Salt in the Elution buffer A. I B. II C. I, III D. II, III E. I, II, III Protein Analysis Gives you information, but are NOT purification techniques Protein Assays: Determines Protein amount (1) General for total protein content • Protein binding Dyes (2) Specific for the protein of interest • • Antibody based assay Absorbance spectroscopy SDS-PAGE: (PolyAcrylamide Gel Electrophoresis) [CH3(CH2)10CH2OSO3–]Na+ Sodium Dodecyl Sulfate intercalates and (SDS)denatures protein and adds negative charge to protein Separates proteins by size: CANNOT be used for purfication, CANNOT recover functional protein. PAGE - larger proteins travel slower so at top of gel; smaller proteins travel faster so at bottom of gel Figure 5-9 Measure Secondary Structure: Indication of folded protein NONE AFTER THIS SLIDE •Circular Dichroism (CD): Sample absorbs right- and leftcircularly polarized light to a different extent measures how much secondary structure is in protein is the protein still functional? Enzyme Activity Assay: Purified protein is active Analyze protein function Experiment: (1) Mix enzyme + substrate (2) Record rate of product formation or reactant disappearance as a function of time – (the rate of reaction) (3) Compare to previous data • New purification [R] • Mutant protein • Different reaction conditions [P] Time Why can you not use SDS-PAGE to purify proteins? A. The SDS denatures the proteins B. It separates protein by size C. You cannot load a sufficient amount of protein on a gel. D. A and C E. You can use SDS-PAGE to purify proteins Determination of Tertiary (and Quaternary) Structure X-Ray Crystallography Nuclear Magnetic Resonance (NMR) Protein Crystals X-Ray Diffraction Patterns can be converted to Electron Density Maps Figure 6-21 Solving the first protein crystal structure (1958) Dickerson, RE; A little ancient history; Protein science, 1992 Nuclear Magnetic Resonance (NMR) Structure determination of molecules in solution •Shows conformational changes/heterogeneity of molecules in solution • size limit (< 50 kDa) Proteins Structures are Dynamic and Flexible (conformational changes are possible) Figure 6-38 Nuclear Overhauser Spectroscopy (NOESY) Spectrum of a Protein Figure 6-24 43 Different Conformations of Brazzein in Solution ...
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