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31 Cards in this Set
- Front
- Back
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What is the mobile phase? |
Where the substances are dissolved in a liquid or gaseous fluid |
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What is the stationary phase? |
Where the disolved mixture is perculated through a coloumn consisting of a porus solid matrix |
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How does the mixture seperate into different bands in chromatography? |
different interactions with the matrix and retarding forces causing migration at different rates |
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How is chromatography classed? |
By its according mobile and stationary phase |
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What happens in ion exchange chromatography? |
Ions that are electrostatically bound to an insoluble and chemically inert matrix are reversibly replaced by ions in solution |
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How is the mixture seperated in ion exchange? |
Different proteins have different affinity for the ion exchanger depending on their net charges |
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The affinity for a protein to an ion exchanger depends on... |
Identity and concentrations of other ions in solution (competition) pH as the net charges vary with pH |
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What is stepwise elution? |
Running the mixture through different concentration salt buffers to seperate |
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What is gradient elution? |
Uses a magnetic stirer to put different concentrations through the column |
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What are molecules seperated due to in gel filtration chromatography? |
Their size and shape |
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What is the basis of gel filtration chromatography? |
The molecules which are too big to enter the pores of the gel beads of the column are eluted first in a smaller elluant volume than the smaller molecues that pass through the gel. |
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What is the exclusion limit? |
The smallest molecule unable to penetrate the pores of a given gel |
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What is the elution volume? |
The volume required to elute the solute from the column |
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What is the void volume? |
The volume of the solvent space surrounding the beads |
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What sis the relative elution volume? |
Charactorises each solute and can be used to estimate molecular masses Ve/Vo |
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What is the basis of affinity chromatography? |
A ligand that binds specifically to a protein of interest is attatched covalently to a resin: when an impure protein solution is passed through the disired protein binds but all otheres are washed away. The desired protein is then released by changing elution conditions. |
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What is reverse phase chromatography used to seperate? |
Used to seperate a mixture of non polar substances. |
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What does the stationary phase consist of in reverse phase chromatography? |
a liquid immobilised on a silica substituted with C8 and C18 alkyl chains |
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What does the mobile phase consist of in reverse phase chromatography? |
A more polar liquid |
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What is the elution phase in reverse phase chromatography? |
A highly non polar liquid which is used to dislodge the substances from the stationary phase |
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How does hydrophobic interaction chromatography seperate by? |
seperates native proteins on the basis of surface hydrophobicity |
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What is the stationary phase in hydrophobic interaction chromatography? |
A hydrophilic substance lightly substituted with hydrophobic chains |
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What is the result of the stationary phase in hydrophobic interaction chromatography? |
Weak interactions which maintain the native fold of the protein |
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How is the protein eluted in hydrophobic interation chromatography? |
Weaking progressively the hydrophobic interactions for example by adding aqueous solutions with decreasing salt conc |
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How is analysis of a fraction eluted from chromatography carried out? |
Electrophoresis |
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Equaition for electrophoretic mobility |
u= v/E= q/F v= rate of migration E=electric field strength q=electric force of an ion F=frictional coefficient |
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How does SDS-Page electrophoresis work? |
SDS unfolds proteins B-mercaptoethanol is used to break S-S linkages SDS causes the proteins to mask their intrinsic charge and move towards + electrode Proteins are seperated on the basis of their molecular masses and are visualised with a dye |
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How can the molecular mass be calculated from SDS-Page electrophoresis? |
The relative mobility of proteins is linearly realated to the logarithm of their molecular mass (5-10% accuracy) |
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What is isoelectric focussing? |
Proteins are seperated electrophoretically on the basis of their native content of acid and base residues The proteins will stop moving when they reach their isoelectric point |
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What is two dimensional gel electrophoresis? |
Isoelectric focussing is combined with SDS-Page electrophoresis They are run in right angles to each other on seperate gels |
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How are proteins detected by immunoblotting? |
Blot proteins from gel onto nitrocellulose Block the unoccupied binding sites on the nitrocellulose with casein Incubate with rabbit antibody to protein of interest Wash and incubare with an enzyme linked goat antibody Assay the linked enzyme with a colormetric reaction |
