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Strategies for protein purification - Coggle Diagram
Strategies for protein purification
Protein purification methods
Affinity chromatography (AC)
Separates proteins on the basis of a reversible interaction between the target protein and a specific ligand attached to a chromatography matrix
Biospecific
Antibodies binding Protein A or a receptor binding a hormaone
Non-biospecific
A protein binding a dye substance or histidine-containing proteins binding metal ions
Offers high selectivity - high resolution - intermediated to high capacity
Elution can often be performed under mild conditions
Can therefore sometime be used for single-step purification
The target protein is specifically and reversibly bound by a complementary binding substance (ligand). The sample is applied under conditions that favor specific binding to the ligand. Unbound material is washed out of the column, and bound target protein is recovered by changing conditions to those favoring elution.
Elusion is performed specifically
Using a competitive ligand
Elusion is performed nonspecifically
By changing pH, ionic strength, or polarity
Often used as a first purification step
Immobilized metal ion affinity chromatography (IMAC)
IMAC is based on the interaction of proteins with histidine residues (or trp and cys) on their surface with divalent metal ions immobilized via a chelating ligand
Ion Exchange Chomatography (IEX)
Separates proteins with differences in surface charge to give high-resolution separation with high sample loading capacity
The separation is based on the reversible interaction between a charged protein and an oppositely charged chromatography medium
Gel Filtration (GF)
(Size-exclusion chromatography)
Simple to use and allows separation of substances with differences in molecular size, under mild conditions
Can be used for protein purification or for group separation in which the samples are separated in two major groups
A non-binding method
No concentration of the samples components take place