Lecture 4 Flashcards
(26 cards)
Protein Purification
The process of selectively eliminating components of a mixture to increase the percentage of a target molecule after each fractionation step.
Salting Out
A purification procedure based on protein solubility, where high salt concentrations lead to protein precipitation due to insufficient water to hydrate protein polar groups.
Salting In
The principle where macroions in low-ionic strength salt solutions experience strong attraction with counterions, increasing solubility.
Ionic Strength (μ)
A measure of the concentration of ions in a solution, calculated by the formula: μ = ½Σcᵢzᵢ², where cᵢ is the concentration and zᵢ is the charge of each ion.
Differential Ammonium Sulphate Precipitation
A method where the solution pH is adjusted to the pI of the target protein, ammonium sulphate is added to increase ionic strength to the protein’s solubility limit, and precipitated contaminant proteins are removed by centrifugation.
Dialysis
A technique for removing salt or performing buffer exchange (changing buffers) using a membrane with a Molecular Weight Cut-off (MWCO). It does NOT concentrate a sample.
Gel Filtration Chromatography
Separates proteins by size using resins/matrices like Sephadex or Sepharose. Larger molecules elute first as they cannot enter the beads, while smaller molecules enter the beads and take longer to elute.
Ion Exchange Chromatography
Separates proteins based on their ionic charge. Proteins bind to charged resins (e.g., Carboxymethyl (CM) group for cation exchange, Diethylaminoethyl (DEAE) group for anion exchange) and are eluted using a gradient of increasing ionic strength.
Hydrophobic Interaction Chromatography (HIC)
Separates proteins based on polarity (hydrophobicity) using hydrophobic matrices like Butyl-sepharose or Phenyl-sepharose. Binding occurs in high ionic strength buffer, and proteins are eluted with a decreasing salt gradient.
Affinity Chromatography
Separates proteins based on specific binding interactions. A ligand (X) is covalently attached to a column matrix, and the target protein binds to it. The target protein is then eluted by adding a high concentration of X. Example: Concanavalin A (glucose-binding protein) using glucose as ligand.
High-Performance Liquid Chromatography (HPLC)
A high-resolution, high-sensitivity, and fast chromatography technique that uses long, narrow columns packed with fine beads and a mobile phase forced through at high pressure. It can separate based on adsorption, ion exchange, size exclusion, HIC, or Reverse Phase Chromatography.
FPLC (Fast Protein Liquid Chromatography)
A type of liquid chromatography often used for protein purification, similar to HPLC but typically operates at lower pressures and uses softer resins.
Gel Electrophoresis
An analytical molecular technique used to separate macromolecules like proteins or DNA based on their physical properties, typically size, charge, and shape, by applying an electric field through a gel matrix.
Agarose Gels
Gels formed by boiling and setting agarose, characterized by larger pore sizes. They are typically used for separating large DNA molecules and are run in horizontal gel electrophoresis setups.
Polyacrylamide Gels
Gels formed by a polymerization reaction, characterized by smaller pore sizes. They are used for separating proteins and small DNA molecules and are typically run in vertical gel electrophoresis setups.
Native Polyacrylamide Gel Electrophoresis (Native PAGE)
An electrophoresis technique where proteins are not denatured, and their quaternary structure can be retained. Proteins migrate based on their mass, charge, and shape, usually run at pH»_space; 9.0 to ensure most proteins are negatively charged.
Sodium Dodecyl Sulfate (SDS)
An anionic detergent used to denature proteins in electrophoresis. SDS binds to proteins, imparting a constant mass-to-charge ratio, causing them to separate primarily based on their molecular weight (Mr).
2-mercaptoethanol
A reducing agent often added to protein samples before SDS-PAGE to break disulfide bonds within and between protein subunits, ensuring complete denaturation.
Discontinuous SDS-PAGE
A gel system composed of two layers: a stacking gel and a resolving (or running) gel, with different compositions (pore sizes and ionic strengths). This setup greatly improves the resolution (separation ability) of the gels by concentrating proteins into a sharp band before they enter the resolving gel.
Stacking Gel (in Discontinuous SDS-PAGE)
The upper gel layer with larger pores (e.g., 5% acrylamide) and lower ionic strength (e.g., 0.125 M Tris-HCl, pH 6.8). It concentrates proteins into a narrow band before they enter the resolving gel.
Resolving (Running) Gel (in Discontinuous SDS-PAGE)
The lower gel layer with smaller pores (e.g., 12% acrylamide) and higher ionic strength (e.g., 0.375 M Tris-HCl, pH 8.8). Proteins are separated by size within this gel.
Role of Glycine in SDS-PAGE
Glycine, a major component of the running buffer, is zwitterionic. Its ionization changes with pH: in the stacking gel (pH 6.8), it moves slower than proteins, creating a leading ion boundary. In the running gel (pH 8.8), it becomes negatively charged and moves faster than proteins, pushing them to stack.
Visualization of Proteins in Gels
After electrophoresis, proteins can be visualized by:
* Staining: Using dyes like SYPRO Red, Coomassie Brilliant Blue (e.g., Coomassie G-250, Simply Blue), or Silver stain (more sensitive than Coomassie).
* Autoradiography: Exposing radioactive gels to X-ray film (most sensitive).
* Western Blot Detection.
Isoelectric Focusing (IEF)
A technique used to separate proteins in an electric field within a pH gradient. Proteins migrate to the point where the pH equals their isoelectric point (pI), at which their net charge is zero.
