protein purification stratigies
TRANSCRIPT
Protein Purification strategies
Purity requirements – Brief guidelines
o Preparation for protein isolationo Protein extraction and solubilizationo Protein concentration determinationo Concentrating protein solution o Ammonium sulfate precipitationo ultrafiltration, Freeze-drying (Lyophilization) , dialysis
o Column chromatography for purification of protein
Steps of protein purification
Three Phase Strategy
Proteins Composition
Separation Principles in Chromatographic Purification
Gel filtrationSeparate by Size
Chromatogram of gel filtration
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Ion Exchange Chromatography
Ion Exchange Type
Ion Exchange titration curveChoice of exchanger group
Test tube method to determine the initial pH
Hydrophobic Interaction Chromatography
Different selectivity of HIC media
Affinity Chromatography
Summary of affinity chromatography
Affinity chromatography is simple to do and can give high purity in one step
Group-specific ligands are the easiest to use
Design the elution scheme
General elution conditions are often harsh
Specific eluents are kinder
Analytical tools
A rapid and reliable assay for the target protein
Purity determination e.g. SDS-PAGE
Total protein determination e.g. colorimetric method
Considerations when linking techniques
Logical Combinations of Techniques
Shortcuts – Rapid establishment ofmilligram scale purification protocols
chromatography computer-controlled system
ÄKTApurifier™ High performance purificationand characterization of proteins
ÄKTApurifier™ High performance purificationand characterization of proteins
Fraction collectors
chromatography computer-controlled system
Fraction Collection
Chromatogram of cation exchange chromatography using SP-sepharose Fast Flow column
0.0
50.0
100.0% Buffer B
1 5 8 12 17 22 27 32 37 42 47 52 57 62 67 72 77 82 87 92 97 103 109 115
-0.010
0.000
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0.040
0.050
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0.070
0.080
0.090
0.100
-1.0
0.0
1.0
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9.0
10.0
00:00:00 01:00:00 02:00:00 03:00:00 04:00:00 05:00:00 06:00:00 07:00:00 08:00:00 09:00:00
Fractions
Hr:Min:Sec mS/cmAU
Chromatogram of hydrophobic interaction chromatography using pheneyl-Sepharose Fast Flow column
SDS-PAGE analysis
Enzyme purification - SDS-PAGE analysis
Prepacked Column
Chromatography Columns
Chromatography Media
Structure of Separation Media
Primary sequence information andamino acid composition from blots (Blotting
to PVDF)
1. Blotting is a technique for the electrophoretic transfer of DNA, RNA or protein to a suitable membrane.
2. For protein sequencing and amino acid analysis, the proteins are transferred to polyvinylidene difluoride (PVDF) membrane.
3. Bands visualized on the blot with Coomassie Brilliant Blue R-250 are excised, cut into smaller pieces, washed extensively with deionized water and used directly for N-terminal sequencing or amino acid analysis
ÄKTApurifier™ High performance purificationand characterization of proteins