[45] Protein Extraction and Purification Flashcards
What is the purpose of protein extraction in molecular biology?
To isolate proteins from biological samples for further study.
What are some common methods of protein extraction?
- Mechanical disruption
- Detergent lysis
- Enzymatic lysis
What is the purpose of a protein purification process?
To isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms.
What techniques are often used in protein purification?
- Precipitation and differential solubilization
- Ultracentrifugation
- Chromatography (Ion exchange, Affinity, Size exclusion)
- Electrophoresis
What factors can influence protein extraction and purification?
- Protein size
- Protein charge
- Protein solubility
- Protein-protein interactions
- Protein-ligand interactions
How can the success of protein extraction and purification be evaluated?
By using assays like SDS-PAGE, Western blotting, or Mass Spectrometry.
What is SDS-PAGE?
Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis, a common method for separating proteins based on their molecular weight.
What is the principle of ultracentrifugation in protein purification?
It separates proteins based on their size and shape by spinning samples at very high speeds.
What is affinity chromatography?
A method of separating biochemical mixtures, based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.
What is ion exchange chromatography?
A process that allows the separation of ions and polar molecules based on their charge.
What is size exclusion chromatography?
It separates proteins based on their size. Larger molecules pass more freely, appearing in the earlier fractions, while smaller molecules get entangled in the stationary phase and appear in later fractions.
What are mechanical disruption methods for protein extraction?
Techniques that physically break down cell walls and membranes, like homogenization, sonication, or bead beating.
What is detergent lysis used for in protein extraction?
It disrupts the lipid bilayer of cells and organelles by solubilizing the membrane proteins and lipids.
What is the purpose of enzymatic lysis in protein extraction?
It uses enzymes like lysozyme or proteases to degrade the cell wall or membrane, freeing the intracellular proteins.
What are proteases?
Enzymes that conduct proteolysis, the breakdown of proteins into smaller polypeptides or single amino acids.
