protein purification stratigies

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

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0.040

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0.080

0.090

0.100

-1.0

0.0

1.0

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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