L10 Electrophoresis Flashcards
(36 cards)
What is electrophoresis?
A technique used to separate charged particles in an electric field.
What is the direction of movement for cations and anions in electrophoresis?
Cations move toward the cathode; anions move toward the anode.
What factors affect the rate of migration in electrophoresis?
Net charge of the molecule, size and shape of the molecule, buffer pH, electric field, and temperature.
Define electrophoretic mobility (μ).
The average velocity (V) of an ion in an applied electric field (E).
What opposes the movement of an ion in an electric field during electrophoresis?
Frictional drag/force (Fd) and electrophoretic retardation force.
How is electric force (Fe) calculated?
Fe = q x E, where q is the charge and E is the electric field strength.
What is the equation for frictional force (Fd)?
Fd = f x v, where f is the frictional coefficient and v is the velocity.
According to Stokes’ law, how is the frictional coefficient (f) calculated?
f = 6πrη, where r is the radius of the molecule and η is the viscosity of the medium.
What occurs when equilibrium is reached during electrophoresis?
The resultant force is zero, and molecules move at a constant velocity.
What is the relationship between mobility (μ) and charge-to-size ratio?
Mobility is proportional to the charge-to-size ratio (μ = q/f).
What is capillary electrophoresis (CE)?
A technique that uses narrow capillaries and high voltages to separate charged molecules.
What is the significance of electroosmotic flow (EOF) in capillary electrophoresis?
EOF results in the bulk movement of fluid near a charged surface when placed in an electric field.
What is used to prepare polyacrylamide gel for one-dimensional gel electrophoresis?
Different concentrations of acrylamide and a cross-linker.
What is SDS-PAGE?
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, used to denature proteins.
What is the role of SDS in SDS-PAGE?
SDS denatures proteins and gives them an overall negative charge.
What is the difference between reducing and non-reducing conditions in SDS-PAGE?
Reducing conditions break disulfide bonds; non-reducing conditions retain them.
What is the purpose of using a gradient gel in electrophoresis?
To allow separation of a greater range of molecular weights.
What does the term isoelectric point (pI) refer to?
The pH at which a protein has no net charge.
What is isoelectric focusing (IEF)?
A technique that separates proteins based on their isoelectric point (pI).
What is the significance of the Ferguson plot?
It is used to determine the molecular weight of proteins in native gels.
What is the limit of detection for Coomassie blue staining?
8-10 ng.
What is the advantage of silver staining in gel electrophoresis?
Can detect as little as 1 ng of protein.
Fill in the blank: The term for the movement of proteins in an electric field until they reach their pI is called __________.
Isoelectric focusing.
True or False: Non-dissociating PAGE retains the three-dimensional conformation of proteins.
True.
