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37 Cards in this Set

  • Front
  • Back

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)

Anion exchangers bind

Negatively chargrd molecules

Cation exchanger bind

Positively charged molecules

The ____ the binding of an individual protein to the ion exchanger, the _____ it will appear in the ____

Stronger


Later


Elution buffer

In pH-gradient chromatography, the pH is changed ____

Continuously or in steps

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

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

A short tail consisting of arginine residues allows a protein to bind a _____ exchanger under conditions where ____

Cation



Almost no other cell proteins bind.

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

Affinity chromatography is based on ____ interactions between ______ and

Highly specific



Immobilized ligand and the protein of interest

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

Examples of Affinity chromatography include purification of ______ which bind to Immobilized _____


(1&2)



1- purification of glycoproteins which bind to Immobilized lectins



2- purification of aerine proteases with lysine binding sites which bind to Immobilized lysine



In Adsorption chromatography, the Stationary phase is ______ polar than the mobile phase

More

In Adsorption chromatography, the protein of interest selectively bind to a _____ matrix under one condition and is released under ______ condition.

Static



Different condition

Adsorption chromatography methods enable

High ratios of product load to Stationary phase volume

The specific binding of antibodies to their epitiposes is used in

Immunoaffinity chromatography

Immunoaffinity chromatography can be applied for the purification of

Either antigen or antibody

In immunoaffinity chromatography, the _____ can be covalently coupled to the Stationary phase and act as the _____ for the _____ to be purified

Antibody



Receeptor



Antigen

In immunoaffinity chromatography, the ____ can be attached to the Stationary phase for the purification of the ____

Antigen



Antibody

Advantages of immunoaffinity chromatography

High specificity



Combination of concentration and purification in one step.

Disadvantage of immunoaffinity chromatography

1- Very strong antibody antigen binding


2- disruption of the covalent bond linking the receptor to the matrix.

In immunoaffinity chromatography, if a disruption of covalent bond linking receptor to the matrix happen, it would result in _____

Elution of the entire complex

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

Examples of proteins of potential therapeutic value that have been purified using immunoaffinity chromatography are

Later

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)

Hydrophobic exposure is suppressed because of _____

The presence of hydrophilic amino acids that attract large clusters of water molecule and form a shield.

High salt concentrations _____ the hydration of a protein, and the surface exposed hydrophobic amino acids residues become ____ accessible

Reduce



More

Hydrophobic interaction chromatography is Based on _____ interactions between ____

Non covalent and non electrostatic interactions between proteins and the stationary phase

_______ separates molecules according to their shape and size

Gel-permeation chromatography


Aka size-exclusion chromatography

Are Inert gels with a narrow pore-size distributions in the size range of proteins available in gel filtration?

Yes

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

The ____ the protein, the more volume it will have available in which to disperse

Smaller

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

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.

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.

Gel-permeation chromatography is usually limited because

Only relatively small sample volumes can be loaded on a large column

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)