BACE Protein Isolation and Quantification 2

Question 1

What is the purpose of a protease inhibitor cocktail added during cell lysis for protein extraction?

Accepted Answer

To prevent endogenous proteases released during cell lysis from degrading the target protein

Answer

To enhance protein solubility by breaking disulfide bonds

Answer

To increase protein yield by improving lysis efficiency

Answer

To precipitate unwanted lipids and nucleic acids from the lysate

Question 2

In SDS-PAGE, why are proteins separated primarily by molecular weight?

Accepted Answer

SDS binds uniformly to proteins (approximately 1.4 g SDS per g protein), giving all proteins a uniform negative charge-to-mass ratio so that migration depends only on molecular size

Answer

SDS gives each protein a unique net charge proportional to its amino acid composition

Answer

The polyacrylamide gel selectively binds large proteins retarding their migration based on charge density

Answer

Reducing agents in the sample buffer separate proteins by charge while SDS separates by size

Question 3

What does a Bradford assay (Coomassie Blue dye binding) measure, and what is its color shift?

Accepted Answer

Total protein concentration; Coomassie Blue shifts from reddish-brown to blue when it binds proteins under acidic conditions

Answer

Total DNA concentration; the dye shifts from blue to clear in the presence of DNA

Answer

Enzyme activity; the blue dye is consumed proportional to enzymatic turnover

Answer

Lipid concentration; the dye shifts from blue to yellow in the presence of membrane lipids

Question 4

What is the principle behind ammonium sulfate precipitation for protein purification?

Accepted Answer

High salt concentrations remove the hydration shell from proteins reducing their solubility until they precipitate (salting out)

Answer

Ammonium sulfate reduces the pH to precipitate proteins at their isoelectric point

Answer

Ammonium sulfate chelates metal cofactors causing metalloenzymes to aggregate and precipitate

Answer

Ammonium sulfate forms covalent crosslinks between adjacent protein molecules causing bulk precipitation

Question 5

In a Western blot procedure, what is the function of the blocking step (e.g., 5% milk or BSA)?

Accepted Answer

To saturate non-specific protein-binding sites on the membrane preventing antibody from binding non-specifically to the membrane background

Answer

To enhance antibody binding to the target protein on the membrane

Answer

To denature the primary antibody so it only detects denatured proteins on the blot

Answer

To transfer blocking agent onto the gel before membrane transfer to improve protein retention

Question 6

What does 'affinity chromatography' exploit to purify a specific protein from a complex mixture?

Accepted Answer

The specific, reversible, non-covalent interaction between the target protein and an immobilized ligand

Answer

Size differences between the target protein and contaminants

Answer

Differences in protein surface charge at a specific pH

Answer

Differences in protein solubility in organic solvents