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

Cells contain proteases that are released during lysis; protease inhibitors block these enzymes, preventing degradation of the protein of interest during extraction.

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 binds proteins at approximately 1.4 g/g, coating them with negative charges proportional to their mass, so all proteins migrate with the same charge-to-mass ratio and are separated only by the sieving effect of the polyacrylamide gel.

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

The Bradford assay measures total protein by the color shift of Coomassie Brilliant Blue G-250 from its reddish-brown (free) form to its blue form upon binding to proteins — measured spectrophotometrically at 595 nm.

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

High concentrations of ammonium sulfate compete with proteins for water molecules, removing their hydration shell; without hydration, protein-protein hydrophobic interactions dominate and they precipitate (salting out).

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

Blocking covers non-specific protein-binding sites on the membrane with irrelevant proteins (milk casein, BSA), preventing primary antibody from sticking non-specifically to the membrane background.

BACE - Biotechnician Assistant Credentialing Exam Practice Test

BACE - Biotechnician Assistant Credentialing Exam Practice Test

BACE Protein Isolation and Quantification 2