Protein Purification BL4010 10.10.05 The basic techniques
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Transcript of Protein Purification BL4010 10.10.05 The basic techniques
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Protein Purification
BL4010 10.10.05
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The basic techniques
• Concentration (size)
– precipitation– ultrafiltration– dialysis– centrifugation
• Chromatography (size/charge/chemistry)
– ion exchange– size exclusion– affinity– hydrophobic interaction
• Electrophoresis (size/charge)
– "native"– denaturing– isoelectric focusing– 2-dimensional
• Immunological– chromatography– in situ imaging– immunoblotting
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Electrophoresis (SDS-PAGE)
• Tris-glycine buffer
• 10% SDS
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Electrophoresis
Protein Normal MW MW with dye Color ovalbumin 45,000 51,000 yellow carbonic anhydrase 29,000 30,000 orange trypsin inhibitor 20,100 23,000 green -lactalbumin 14,200 16,500 purple aprotinin 6,500 10,500 blue
“Prestained” markers have dyes covalently bound BEFORE electrophoresis - increased MW
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Electrophoresis
• Protein detection using dyes– Coomassie blue– Sypro– Cybergreen– Silver staining
coomassie brilliant blue A595
Staining with dyes AFTER electrophoresis - no change in MWnon-covalent interaction
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Western blotting
• Separate proteins by electrophoresis
• Transfer to membrane (e.g. nitrocellulose)
• Bind primary antibody• Bind secondary
antibody• Detection
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Immuno-Affinity Chromatography
• antibody fixed to matrix
• protein binds to antibody
• wash unbound and loosely bound proteins off column
• elute protein with change in salt/pH
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Hydrophobic interaction chromatography
• Hydrophobic group bound to solid phase• Binding
– high salt (increases water surface tension, decreases available water molecules, increases hydrophobic interactions)
• Elution– decrease salt – add detergent– decrease polarity of mobile phase
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Assay and Specific Activity Fraction Volume
(ml)Total
protein (mg)
Total activity
Specific Activity
(activ./mg)
Percent Recovery
(ratio t.a.)
Fold
Purificat'n
(ratio s.a.)
Crude extract
3,800 22,800 2460 0.108 100 0
Salt ppt. 165 2,800 1190 0.425 48 3.9
IEC 65 100 720 7.2 29 66
SEC 40 14.5 555 38.3 23 355
Affinity 6 1.8 275 152 11 1407
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Criteria for purity
When is protein pure or pure enough?
• homogeneity– protein complexes?
• constant specific activity
• Practical: further attempts at purification are futile since the only material left in the fraction is the material that actually is responsible for the activity being assayed.
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Protein purification simuation
• http://www.tlsu.leeds.ac.uk/courses/bioc2060/proteinlab102/proteinlab.html
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Enzymes
BL4010 10.12.05
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• What is an enzyme?
• How do enzymes work?– energetics– underlying general mechanism– components (prosthetic groups, coenzymes)– specific mechanisms
Ch.13.1, 13.2, 14.1, 14.2, 14.3, 14.4, 14.5
Objectives
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What is an enzyme?
Macromolecular biological catalyst
Can be protein or RNA
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What is an enzyme?
Macromolecular biological catalyst
What is a catalyst?– is not altered by reaction
• participates but emerges unchanged
– increases the rate at which substrates and products reach equilibrium
– does not alter equilibrium
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Why enzymes?
• Why invest energy and resources into creating a large catalyst?– Enzymes endow cells with the remarkable
capacity to exert kinetic control over thermodynamic potentiality
• Fine tune selectivity (substrate binding specificity)• Fine tune catalytic rate• Additional regulatory control (e.g. allostery,
signalling networks)
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Enzymes are good catalysts
• Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates!
• Urease is a good example: – Catalyzed rate: 3x104/sec
– Uncatalyzed rate: 3x10 -10/sec
– Ratio is 1x1014 !
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Enzymes are selective catalysts
• Enzymes selectively recognize proper substrates over other molecules
• Enzymes produce products in very high yields - often much greater than 95%
• Specificity is controlled by structure - the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield
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How do enzymes work?• How do catalysts in general work?
catalysts lower the activationenergy of a reaction
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The transition state
Understand the difference between G and G‡
• The overall free energy change for a reaction is related to the equilibrium constant
• The free energy of activation for a reaction is related to the rate constant
• It is extremely important to appreciate this distinction!
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How do enzymes work?
• Enzymes accelerate reactions by lowering the free energy of activation
HOW?
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Four contributing factors to enzyme catalysisNO ONE MECHANISM ACCOUNTS FOR CATALYSIS ALONE!
• Specific substrate binding– local concentration of reactants– productive orientation of reactants– binding energy used to offset loss of entropy
• Control over solvent interactions– desolvation (binding energy offsets)– ordered solvent in binding pocket
• Induction of strain on reactants• Alternate reactive pathway
– transient involvement of enzyme functional groups
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How do enzymes work?
• Enzymes accelerate reactions by lowering the free energy of activation
• Enzymes do this by binding the transition state of the reaction better than the substrate