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The basic techniques. Concentration (size)precipitationultrafiltrationdialysiscentrifugationChromatography (size/charge/chemistry)ion exchangesize exclusionaffinityhydrophobic interaction. Electrophoresis (size/charge)"native"denaturingisoelectric focusing2-dimensionalImmunologicalchromatographyin situ imagingimmunoblotting.
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1. Protein Purification BL4010 10.10.05
2. 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
3. Electrophoresis (SDS-PAGE) Tris-glycine buffer
10% SDS
4. Electrophoresis
5. Electrophoresis Protein detection using dyes
Coomassie blue
Sypro
Cybergreen
Silver staining
6. Western blotting Separate proteins by electrophoresis
Transfer to membrane (e.g. nitrocellulose)
Bind primary antibody
Bind secondary antibody
Detection
7. 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
8. 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
9. Assay and Specific Activity
10. 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.
11. Protein purification simuation http://www.tlsu.leeds.ac.uk/courses/bioc2060/proteinlab102/proteinlab.html
12. Enzymes BL4010 10.12.05
13. 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
14. What is an enzyme? Macromolecular biological catalyst
Can be protein or RNA
15. 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
16. 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)
17. 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 !
18. 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
19. How do enzymes work? How do catalysts in general work?
20. 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!
22. How do enzymes work? Enzymes accelerate reactions by lowering the free energy of activation
HOW?
23. 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
24. 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