Electrophoresis and Chromatography Flashcards
What is electrophoresis?
Movement of charged particles in a fluid under a uniform electric field. Molecules move at a constant velocity proportional to their charge density.
What is Polyacrylamide Gel Electrophoresis (PAGE)?
Separates proteins based on size, shape, and charge density. Large, globular proteins move slower than small, elongated ones.
What is SDS PAGE?
PAGE with added dodecyl sulfate (SDS) to proteins. SDS binds to amino acid residues (1:2 ratio) to impart a uniform negative charge and unfold proteins. Separates proteins based only on size.
What role does β-mercaptoethanol play in SDS PAGE?
Reduces disulfide bonds, further denaturing proteins.
What is the difference between stacking and running gels in SDS PAGE?
Stacking gel (low polyacrylamide) concentrates proteins into thin bands. Running gel (high polyacrylamide) separates proteins by size due to the gel’s sieving effect.
How can proteins be stained in SDS PAGE?
Silver stain: 10-100x more sensitive. Coomassie blue: Less expensive but less sensitive.
How is protein size determined in SDS PAGE?
Migration distance is inversely proportional to the protein’s size logarithm in a linear range.
What is Native PAGE?
Preserves the native structure of proteins. Separates proteins based on size, charge, and conformation.
How does SDS PAGE differ from Native PAGE?
SDS PAGE denatures proteins, separating them by size. Native PAGE preserves native protein structure, separating based on size, charge, and conformation.
What is IEF gel?
Polyacrylamide gel with a pH gradient created by carrier ampholytes. Proteins are separated based on their isoelectric points (pI).
How does IEF gel work?
Low pH: Proteins have a positive charge (migrate toward negative electrode). High pH: Proteins have a negative charge (migrate toward positive electrode). When a protein reaches its isoelectric point (pI), its net charge becomes zero, and migration stops.
What is 2D PAGE?
Combines IEF (separation by pI) and PAGE (separation by size). First dimension: Isoelectric point (charge). Second dimension: Size (via PAGE).
What is chromatography?
A technique where molecules are separated as they move through a stationary phase while a mobile phase moves them.
What is Low-Pressure Liquid Chromatography (LPLC)?
Solutions flow under gravity; no pressure is applied.
What is High-Performance Liquid Chromatography (HPLC)?
Uses fine matrix beads for higher interaction sites, requiring high pressure to maintain a fast flow rate.
What are the types of protein chromatography?
Size-exclusion chromatography (SEC), Ion exchange chromatography, Affinity chromatography, Reversed-phase chromatography.
How does Size-Exclusion Chromatography (SEC) work?
Smaller proteins enter pores in beads, while larger proteins pass around beads and elute faster. Unlike SDS PAGE, larger proteins travel faster.
How does Ion Exchange Chromatography work?
Proteins are separated based on their net electrical charge (sum of charges from amino acid residues). pH < pI: Protein has a positive net charge. pH > pI: Protein has a negative net charge. The stationary phase is beads covalently linked to charged groups.
How does Affinity Chromatography work?
Uses the specific binding affinity of proteins to a target molecule. The stationary phase has beads covalently linked to the target molecule that the protein specifically binds to.
How does Reversed-Phase Chromatography work?
A non-polar matrix forms hydrophobic interactions with proteins. Proteins are separated by their hydrophobicity. The matrix is typically silica with chemically active alkyl groups. Proteins elute with an organic solvent (e.g., acetonitrile) disrupting hydrophobic interactions.
What is the summary of chromatography types?
Gel Filtration: Separates by size. Ion Exchange: Separates by charge. Affinity: Separates by specific interaction. Reversed-Phase: Separates by hydrophobicity.
Can chromatography be performed using LPLC or HPLC?
Yes, all chromatography types (SEC, Ion Exchange, Affinity, and Reversed Phase) can be performed under both LPLC and HPLC conditions.
