Term
| one way to study proteins |
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Definition
| purify them and study them in vitro |
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Term
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Definition
the functional representation of the genome
encompassesd the types, functions, and interactions of proteins that yield a functional unit |
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Term
| something the proteome tells us |
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Definition
| what proteins are functionally present |
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Term
| how proteins interact in the body |
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Definition
| interact with each other to form complexes with specific functional properties |
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Term
| how we acquire an understanding of the proteome |
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Definition
| by isolating, characterizing, and cataloging proteins |
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Term
| the first step in understanding a protein's function |
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Definition
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Term
| proteins can be purified based on... |
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Definition
| their chemical properties |
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Term
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Definition
| procedure to measure the activity of a biomolecule, such as an enzyme |
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Term
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Definition
| the ratio of enzyme activity to the amount of protein in the enzyme assay at the end of each step of purification |
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Term
| information you need in order to make sure the protein purification scheme is working |
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Definition
-enzyme activity
-amount of total protein in mixture |
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Term
| the fractions that result from centrifugation |
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Definition
-pellet
-supernatant
called fractions because we fractionate the homogenate cell contents |
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Term
| differential centrifugation |
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Definition
putting the supernatant thru increasingly forceful rounds of centrifugation
yields pellets of decreasing density |
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Term
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Definition
| the fraction that is used as a source for further purification |
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Term
| properties by which proteins can be purified |
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Definition
-solubility
-size
-charge
-specific binding affinity |
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Term
| some separation mechanisms that can be used to purify a protein |
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Definition
-salting out
-separation by size
-ion-exchange chromatography
-affinity chromatography
-high-pressure liquid chromatography |
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Term
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Definition
| increase in salt concentration to make a protein dissolve in solution |
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Term
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Definition
| protein precipitation caused by increasing salt concentration |
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Term
| salt concentration vs. protein solubility |
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Definition
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Term
| disadvantage of salting out |
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Definition
| can make the protein lose its function |
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Term
| what you gotta do to after salting out a protein |
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Definition
| put the solution thru dialysis |
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Term
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Definition
the protein-salt solution is closed in a semipermeable bag which itself is immersed in a solution of low salt concentration (the dialysate)
small molecules diffuse from the bag |
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Term
| molecular exclusion chromatography aka gel-filtration chromatography |
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Definition
| separates proteins on the basis of size |
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Term
| how molecular exclusion chromatography aka gel-filtration chromatography is done |
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Definition
| percolating mix of proteins thru column of beads that increase in size as you go down the column |
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Term
| ion-exchange chromatography |
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Definition
| separating proteins on the basis of their net charge |
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Term
| how ion-exchange chromatography is done |
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Definition
| percolating mix of proteins thru column of beads that contain charged groups |
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Term
| how salting out precipitates out proteins |
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Definition
| by competition between the salt ions and the protein to keep the ion in solution |
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Term
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Definition
| separates proteins by their affinity for certain substances |
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Term
| high-pressure liquid chromatography |
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Definition
| similar to the other chromatography techniques, but uses much finer beads and high pressure is used |
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Term
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Definition
| a molecule with a net charge moving in an electric field |
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Term
| how gel electrophoresis is done |
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Definition
| proteins move thru the gel, the speed and distance depending on molecular weight and charge |
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Term
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Definition
| type of electrophoresis that uses SDS to linearize the proteins and "swamp" the charges on them, such that they differ only in size |
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Term
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Definition
| separating proteins electrophoretically based on the relative content of acidic and basic residues of the proteins |
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Term
| how isoelectric focusing is done |
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Definition
-mix of proteins undergoes electrophoresis in a pH gradient
-each protein migrates in the gel until it reaches pI |
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Term
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Definition
isoelectric point
this is the pH at which a protein has no net charge |
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Term
| two-dimensional electrophoresis |
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Definition
| first putting proteins thru isoelectric focusing, then putting them thru SDS-PAGE in a perpendicular direction |
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Term
| how the success of a protein purification scheme is determined |
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Definition
| by determining specific activity and performing an SDS-PAGE analysis |
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Term
| parameters that are measured when purifying a protein |
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Definition
-total protein
-total activity
-specific activity
-yield
-purification level |
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Term
| how the quantity of protein in a fraction is measured |
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Definition
| quantity = protein c'tration X volume |
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Term
| how the enzyme activity for a fraction is obtained |
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Definition
| total enzyme activity = enzyme activity in fraction X volume of fraction |
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Term
| how specific activity is obtained |
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Definition
| specific activity = total activity / total protein |
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Term
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Definition
measured as the total activity retained after each purification step as a percentage of the activity in the crude extract
activity in initial extract = 100% |
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Term
| how purification level is measured |
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Definition
| purification level = specific activity / specific activity of initial extract |
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