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37 Cards in this Set
- Front
- Back
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In ion exchange chromatography, the type of column needed is determined by |
The properties of the proteins to be purified. ( isoelectric poibt and charge density) |
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Anion exchangers bind |
Negatively chargrd molecules |
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Cation exchanger bind |
Positively charged molecules |
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The ____ the binding of an individual protein to the ion exchanger, the _____ it will appear in the ____ |
Stronger Later Elution buffer |
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In pH-gradient chromatography, the pH is changed ____ |
Continuously or in steps |
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In pH-gradient chromatography, the protein binds at 1 pH and is releases at ______ pH. As a result of the heterogeneity in glycosylation, glycosylated proteins may elute in a relatively _____ pH range |
Different Broad |
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In order to simplify purification, |
A specific amino acid tail can be added to the protein at the gene level to create a purification handle |
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A short tail consisting of arginine residues allows a protein to bind a _____ exchanger under conditions where ____ |
Cation Almost no other cell proteins bind. |
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Purification handle technique is only useful for _____, and cannot be used for ______ due to _____ |
Laboratory scale isolation Can't be used for production scale due to regulatory problems related to the removal of the arginine or other specific c tags from the protein |
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Affinity chromatography is based on ____ interactions between ______ and |
Highly specific Immobilized ligand and the protein of interest |
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In Affinity chromatography, ______ of this matrix will remove contaminants, and the purified protein can be recovered by ____ competing for the ______ phase binding sites or by ______ which greatly reduces Affinity |
Extensive washing
Addition of ligands
Stationary phase
Changes in physical conditions |
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Examples of Affinity chromatography include purification of ______ which bind to Immobilized _____ (1&2)
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1- purification of glycoproteins which bind to Immobilized lectins
2- purification of aerine proteases with lysine binding sites which bind to Immobilized lysine
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In Adsorption chromatography, the Stationary phase is ______ polar than the mobile phase |
More |
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In Adsorption chromatography, the protein of interest selectively bind to a _____ matrix under one condition and is released under ______ condition. |
Static
Different condition |
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Adsorption chromatography methods enable |
High ratios of product load to Stationary phase volume |
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The specific binding of antibodies to their epitiposes is used in |
Immunoaffinity chromatography |
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Immunoaffinity chromatography can be applied for the purification of |
Either antigen or antibody |
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In immunoaffinity chromatography, the _____ can be covalently coupled to the Stationary phase and act as the _____ for the _____ to be purified |
Antibody
Receeptor
Antigen |
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In immunoaffinity chromatography, the ____ can be attached to the Stationary phase for the purification of the ____ |
Antigen Antibody |
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Advantages of immunoaffinity chromatography |
High specificity Combination of concentration and purification in one step. |
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Disadvantage of immunoaffinity chromatography |
1- Very strong antibody antigen binding 2- disruption of the covalent bond linking the receptor to the matrix. |
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In immunoaffinity chromatography, if a disruption of covalent bond linking receptor to the matrix happen, it would result in _____ |
Elution of the entire complex |
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Why is it always necessary to make a further purification step after Affinity chromatography? |
To make sure there there is no Elution of the complex |
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Examples of proteins of potential therapeutic value that have been purified using immunoaffinity chromatography are |
Later |
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Under physiological conditions, most ______ amino acid residues are located inside the protein core, and only a small fraction of ____ amino acids is exposed in the surface of a protein |
Hydrophobic (1&2) |
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Hydrophobic exposure is suppressed because of _____ |
The presence of hydrophilic amino acids that attract large clusters of water molecule and form a shield. |
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High salt concentrations _____ the hydration of a protein, and the surface exposed hydrophobic amino acids residues become ____ accessible |
Reduce More |
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Hydrophobic interaction chromatography is Based on _____ interactions between ____ |
Non covalent and non electrostatic interactions between proteins and the stationary phase |
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_______ separates molecules according to their shape and size |
Gel-permeation chromatography Aka size-exclusion chromatography |
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Are Inert gels with a narrow pore-size distributions in the size range of proteins available in gel filtration? |
Yes |
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Inert gels (with narrow pore size distribution etc) are packed into a column and the protein mixture is then loaded _____ of the column and the proteins ____ |
On top Diffuse into the gel |
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The ____ the protein, the more volume it will have available in which to disperse |
Smaller |
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Molecules that are larger than the largest pores are not able to penetrate the gel beads, and will therefore ___ |
Stay in the void volume of the column |
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When a continuous flow of buffer passrs through the column, the _____ will elute first and _____ last |
The larger proteins will elute first and the smallest molecules last. |
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Gel-permeation chromatography is a good as an alternative for |
An alternative to membrane diafiltration for buffer exchange at Almost any purification stage, and is often used in laboratory design. |
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Gel-permeation chromatography is usually limited because |
Only relatively small sample volumes can be loaded on a large column |
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Gel-permeation chromatography is commonly used as the ___ step in the purification to bring proteins in the appropriate ____ |
Final step Appropriate buffer used in the final formulation (in this application it has little or no effect on the product purity characteristics) |
