Protein Purification BITC1402 Biotech Methods Loomis-Price

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Protein Purification

• BITC1402
• Biotech Methods
• Loomis-Price

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Getting it Right the First Time
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Goals for Bioseparation Strategies
Yield
Chosen Strategy
Cost
Purity
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Biological Product Flow-Chart
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Assays

• General
• Specific

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Lysis - Physical
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Lysis - Chemical
Organic solvent
Detergent
Free Proteins
Lysozyme
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Keeping Proteins Happy
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Biological Buffers
HEPES
http//www.emdbiosciences.com/docs/docs/LIT/Buffer
s_CB0052_E.pdf
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Protease Inhibitors
http//www.piercenet.com/Objects/View.cfm?typePro
ductFamilyID02040806
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Keep Biological Molecules Cold!
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Chemistry Interlude
For every 10 degrees cooler, degradation occurs
half as fast.
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Changing Buffers - dialysis
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Changing Buffers Filters
Centrifuge microfilter
Ultrafiltration cell (Millipore)
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Removing buffers
Speed-vac (Savant)
Lyophilization (freeze-drying)
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How lyophilization works
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Volatile Buffers

In water
Low Pressure

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Purification Steps
Column Chromatography
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Centrifugation
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Centrifuge Nomogram
http//aquaticpath.umd.edu/nomogram.html
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Salting Out
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Column Chromatography
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Chromatography Matrix
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Chromatography Matrix Gel Filtration
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Chromatography Matrix Ion Exchange
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Chromatography Matrix Affinity
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Determining Purity - Gels
SDS-PAGE
Isoelectric focusing
2-D gels
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Determining Purity - Immunoassay