Purification, detection, and characterization of proteins

Question 1

3 Most Widely Used Characteristics For Separating Proteins

Answer 1

Size, defined as length or mass; net electrical charge; and affinity for bind to specific ligands

Question 2

Centrifugation

Answer 2

Two types of particles in suspension with different masses or densities will settle to the bottom of a test tube at different rates

Question 3

Centrifuge

Answer 3

Speeds sedimentation by subjecting particles in suspension to centrifugal forces as great as 1 million times the force of gravity

Question 4

Centrifugation Purposes

Answer 4

1. As a preparative technique to separate one type of material from others with the goal of obtaining enough of the material to perform subsequent experiments
2. As an analytical technique to measure physical properties (e.g. molecular weight, density, shape, and equilibrium-binding constants) of macromolecules

Question 5

Differential Centrifugation

Answer 5

Separates water-soluble proteins from insoluble cellular material:
1. Cell homogenate is poured into a tube
2. Tube is spun at rotor speed to force cell organelles, large unbroken cells, or large cell fragments to collect as a pellet at the bottom
3. Supernatant (liquid full of soluble proteins) is poured off

Question 6

Density Gradient

Answer 6

Solution of increasing density; concentrated sucrose solution is commonly used to form a density gradient in a centrifuge tube

Question 7

Rate-Zonal Centrifugation

Answer 7

1. Protein-mixture is placed on top of a sucrose density gradient in a tube and subjected to centrifugation
2. Each protein in the mixture migrates down the tube at a rate controlled by the protein’s physical properties
3. Proteins start at the thin layer of the sample placed at the top of the tube and separate into bands (zones) of proteins of different masses as they travel at different rates through the gradient

Question 8

Electrophoresis

Answer 8

Any of several techniques for separating macromolecules based on their migration in a gel or other medium subjected to strong electric field