Lecture 6 Working with proteins

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Lecture 6Working with proteins
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Protein purification

• Source of protein
• Overexpression of protein
• Fractionation procedures
• Solubility
• Salting out ((NH4)2SO4)
• pH changes
• Size
• Centrifugation
• Size exclusion (gel filtration)
• Charge/polarity
• Ion exchange chromatography
• Hydrophobic exchange chromatography
• Other
• Affinity chromatography

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Size-exclusion Chromatography
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Separation base on size
1. Zonal centrifugation
Based on sedimentation coefficient (s)
m mass of protein (1-v?) buoyancy factor
m(1-v?)
s
f
f frictional coefficient
Spin in centrifuge
Separation according to sedimentation coefficient
Layer sample on top
Tube with density gradient
Particles of higher M.W and density will be
closer to bottom of the tube
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Ion Exchange Chromatography

• Example DEAE - Cellulose
• (Diethylamino Ethyl)

Anion exchange() charged resin, binds (-)
proteins Cation exchange(-) charged resin,
binds () proteins
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Ion Exchange Chromatography
1.Bind protein to column in low salt
buffer 2.Wash column 3.Elute protein with high
salt buffer.
Fractions
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Ion Exchange Chromatography
Salt
Protein
Activity
2
4
6
8
10
12
Fraction
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Affinity Chromatography
See Fig 6-2 (b)
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Polyhistidine tag purification
Genetically modified protein with 6 or more
histidines at C or N-terminal end
Nickel-containing column
Elute with imidazole
Pure protein with imidazole
(Can be removed by dialysis)
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Gel electrophoresis
m electrophoretic mobility
?velocity of particle
z
?
Eelectric field strength znet charge on
protein ffrictional coefficient

m
E
f
Net charge on protein comes from SDS (negatively
charged detergent)
Proteins are separated on the basis of mass
(frictional coefficient) SDS disrupts almost all
noncovalent interactions SDS binds to protein 1
SDS molecule per 2 amino acids
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Gel electrophoresis
Polyacrylamide gel formation
O
H
)
(
NH
CH2
C
C
H2C
2

acrylamide
methylenebisacrylamide
S2O82-
SO4- sulfate free radical
H
H
H
C
H2C
C
C
H2C
H2C
Pore size can be controlled by adjusting the
concentration of the monomer and crosslinker
CO
NH
CO
NH2
CO
NH2
CH2
CO
NH
H
H
C
H2C
C
C
H2C
H2C
H
CO
NH2
CO
NH2
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Electrophoresis
Log(MW)
Relative mobility
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Analytical ultracentrifugation
Same principle as zonal centrifugation Spin at a
low speed until sedimentation rate is
counterbalanced by diffusion rate
Smooth gradient of protein develops Measure
conc.(c1,c2) at different points(r1,r2) from
center of rotor
Very precise way to determine MW
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Isoelectric focusing
-
Decreasing pH
(stable pH gradient)

Apply protein to top
Apply electric field protein migrates to pH
where its net charge is zero (pI)