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Protein Purification
Protein Purification and Detection
| Question | Answer |
|---|---|
| Protein Therapeutics vs. Small Molecule | Contain intrinsic infectous agents, aseptic techniques required during production, usually have heterogenou composition, exact structure may be unknown |
| Name expected things of proteins | Size, charge, hydrophobicity, correct folding (S-S bonds), subunits, glycosylation, bioactivity |
| Name unexpected things of proteins | Aggregation, incorrect folding, amino acid modifications: Oxidation, Deamination, Cys |
| Biuret Method of Protein Quantification | Copper reacts with peptides or proteins at alkaline pH. Protein is quantified by UV absorbance at 540nm. Requires large quantities for detection. Sensitive to nitrogen-containing substances (leads to error) |
| Lowry Test of Protein Quantificaiton | Based on peptide bond can bind copper. Most of the reactivity due to tyr and trp groups. Only need a lil protein. Blue-green color from reactions. UV at 750nm. |
| Bradford Method of Protein Quantification | Very sensitive/needs low conc. Can bind detergents. Forms blue complex when bound to + or hydrophobic chains of protein. UV at 595nm. |
| Name disadvantages of Bradford Method | Surfactants interfere. Greater variability in response of proteins |
| If you have an OD280 what is the approximate protein concentration? | 0.6mg/ml |
| Fractionation by centrifugation (Purification) | Centriguge at different speeds. Nuclei will come down at diff speed than mitochondria and etc. Zonal Centrifugation: Separate them by density |
| Fractionation by solubility (Purification) | The use of salt can be used to selectively precipitate some proteins while others remain in solution. pH and temperature changes can also alter solubility |
| Column Chromatography: Ion Exchange (Protein Purification) | Column matrix is charged (either - or +) The weakest interacting proteins elute first based on binding ability. At a pH below the pI protein will have a positive charge. At neutral pH almost all - charged. |
| Column Chromatography: Size Exclusion | Column matrix contains pores of specific size. Smaller proteins enter the pore and take longer to come down. Larger ones migrate fastest through column. |
| Column Chromatography: Hydrophobic Interaction | Column matrix binds proteins with exposed hydrophobic surfaces. Hydrophobic interaction is the strongest when ionic strength is really high. High salt promotes hydro interactions. |
| Column Chromatography: Affinity | Column matrix has covalently attached chemical group that proteins of interest bind to. Protein is eluted by adding excess chemical that will compete for binding to protein. Glutathione S-transferase has a high affinity with GSH. |
| SDS page (Purity and Molecular Weight) | SDS is added to protein sample so that all proteins are denatured and possess a - charge. Each protein posses a similar charge-to mass ratio. Larger proteins move slower in gel. A set of MW markers are used so that the MW of unknown can be established. |
| Isoelectric Focusing | A pH gradient is established within a gel and proteins added will migrate through the gradient until they reach a pH that matches their pI. |
| 2D Gel | Takes advantage of differences in pI and MW. Uses isoelectric focusing and then SDS page. Checks purity and specific activity. |
| Can we tell if a protein was deaminated using SDS page? | No we wouldn't be able to b/c it is the same size, we would need isoelectric focusing b/c the pH will be lower which causes a lower pI. |
| Immunoaffinity Chromatography | Antidbody bound covalently to column. Antibody immobilized and can put down the antigen. |
Created by:
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