Term
| First step in purifying a protein |
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Definition
To get the protein out of the cell
-Solubilization/Stabilization
-Choose best source
-Must have method to detect protein of interest |
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Term
| Second step in purifying a protein |
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Definition
| Use fractionation steps to separate protein of interest from all other proteins |
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Term
| Last step in purifying a protein |
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Definition
| Visualize the protein to confirm purity |
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Term
| What you want to know in protein purification |
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Definition
1. How much total protein is present
2. How much of desired protein is present
3. How pure |
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Term
| Detection of proteins and amino acids |
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Definition
Colormetric- uses dyes like coomassie blue
UV
*Remember Phe, Tyr, and Trp absorb UV*
Absorbance at 280 nm is a good diagnostic device for proteins |
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Term
Purification Assessment
-At each separation step |
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Definition
Quantitate total protein concentration
Desired Protein Concentration
If an enzyme, calculate enzymatic activity |
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Term
| Most Separation Techniques based on what? |
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Definition
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Term
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Definition
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Term
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Definition
Ion exchange chromotography Electrophoresis
Isoelectric focusing |
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Term
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Definition
| Hydrophobic interaction chromotography |
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Term
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Definition
Gel filtration chromotography
SDS-PAGE |
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Definition
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Term
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Definition
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Term
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Definition
-Initially salts reduce the electrostatic interactions between proteins increasing solubility
-However eventually the salts compete with the proteins for interactions with water causing the proteins to precipitate |
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Term
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Definition
Most fractionation techniques involve column chromotography
Mobile Phase
-Protein soln
Stationary phase
Eluent
Effluent |
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Term
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Definition
| Another name for the soln that is added to the column |
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Term
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Definition
| Is another name of what comes out of the column |
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Term
| Gel Filtration or Size Exclusion |
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Definition
| Separate by Molecular Weight |
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Term
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Definition
Separate according to charge
-Positively charged protein binds to negatively charged bead
-Negatively charged protein flows through |
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Term
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Definition
Centrifugation
Fractionation |
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Term
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Definition
| Separate according to binding affinity |
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Term
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Definition
Estimate Molecular weight and purity
SDS-detergent used to denature protein and disrupt interactions
All proteins coated with SDS have negative charge
Thus separate by size rather than charge |
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Term
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Definition
Used to determine pI of protein
1D gel
-pH gradient is established in gel
-Protein is applied
-Migrates toward either + or - until it reaches pH where the protein has no net charge |
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Term
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Definition
1st do 1D Isolecetric gel
-separate according to pI
2nd apply 1D gel to SDS gel
-Know separated according to mW |
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Term
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Definition
Separate Subunits
Break Disulfide Bonds
End group analysis
Cut protein
Cut protein again
Reconstruct sequence |
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Term
| Amino acid composition analyzed by |
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Definition
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Term
| Separation of polypeptide chain achieved by |
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Definition
extreme pH
8M guanidine
HCl or high salt concentration |
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Term
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Definition
| Dansyl Chloride, FDNB or similar reagent reacts with primary amines |
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Term
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Definition
| Carboxypeptidase or other exopeptidases |
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Definition
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Definition
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Definition
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Definition
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Definition
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Term
| Sequence all the peptides produced |
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Definition
Usually by Edman degradation
Edman's reagent- phenylisothicyanate |
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