Protein Separation

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Protein Separation

• Lecture 16

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• How do you purify a single recombinant protein of
  interest from over 4,000 naturally occuring E.
  coli gene products.

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Separation Techniques

• Sedimentation
• Chromatography

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Sedimentation
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Sedimentation
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Chromatography

• Chromatography (from Greek ???µachroma, colour
  and ??afe??"grafein" to write) is the collective
  term for a family of laboratory techniques for
  the separation of mixtures. It involves passing a
  mixture dissolved in a "mobile phase" through a
  stationary phase, which separates the analyte to
  be measured from other molecules in the mixture
  and allows it to be isolated.

http//en.wikipedia.org/wiki/Chromatography
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• Separation of Color

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Types of Protein Chromatography

• Size Exclusion Chromatography
• Ion-Exchange Chromatography
• Affinity Chromatography
• Hydrophobic Interaction Chromatography

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Size Exclusion Chromatography
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Ion-Exchange Chromatography
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Affinity Chromatography
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Hydrophobic Interaction Chromatography
Hydrophobic bead

• Aqueous solution hydrophilic
• High salt hydrophobic

H
O
-
-
H

O
O
S
N
H
H
O
O
-
O
S
O
-
O
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Step 1 Add Cell lysate with low salt buffer
Hydrophobic Interaction Chromatography
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Step 2 Wash Column with high salt buffer
Hydrophobic Interaction Chromatography
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Hydrophobic Interaction Chromatography

• Elute GFP from column by adding no salt buffer
• GFP
• Released from column matrix
• Flows through the column