Isolating and purifying proteins

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• Isolating and purifying proteins
• Protein purification
• Molecular weight determination
• Amino acid composition.

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• 1.Methods for purification
• Ammonium sulfate precipitation
• Salting out.
• B. Column chromatography
• Gel filtration
• Ion exchange
• Affinity chromatography

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(NH4)2SO2 Precipitation
Mixture of proteins
Add low concentration of (NH4)2SO2
Precipitates out
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You are left with
Add more
(NH4)2SO2
precipitates out.
is left in the supernatant.
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B. Chromatography
Column chromatography
Taken from Campbell Farrell
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Moving phase Stationary phase
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Small
1. Gel filtation
Large
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Mixture
Beads with holes in them.
Small molecules enter beads. Large molecules
stay outside.
Large molecules come through column first.
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2. Affinity chromatography
Enzymes bind substrate specifically.
E
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1. Bead binds the substrae
2. Add mixture of protein molecules

• Only the enzyme can bind to the
• substrate on the bead.

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4. Add excess substrate and the enzyme comes off
the bead.
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Add mixture of proteins with and - charges
Add salt, comes off
3. Ion exchange

Beads with negative charges.
Protein mix
Binds
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-
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2. Determining the molecular weight of a
protein. A. SDS gel electrophoresis B. Mass
spectroscopy
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A. SDS gel electrophoresis
How do you determine the size (Molecular weight)
of a protein? SDS-gel electrophoresis
Sodium dodecyl sulfate
Negatively charaged.
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Hydrophobic
Amphipathic molecule
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Protein SDS
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Protein SDS
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SDS
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Start
Small
Large
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