Protein Isolation and Quantification Flashcards
6 cards from real BACE practice questions. Tap to flip, then mark Knew It or Still Learning — missed cards come back until you master them.
Read the first 6 Protein Isolation and Quantification flashcards as text
What is the purpose of a protease inhibitor cocktail added during cell lysis for protein extraction?
Answer: To prevent endogenous proteases released during cell lysis from degrading the target protein
Cells contain proteases that are released during lysis; protease inhibitors block these enzymes, preventing degradation of the protein of interest during extraction.
In SDS-PAGE, why are proteins separated primarily by molecular weight?
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
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.
What does a Bradford assay (Coomassie Blue dye binding) measure, and what is its color shift?
Answer: Total protein concentration; Coomassie Blue shifts from reddish-brown to blue when it binds proteins under acidic conditions
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.
What is the principle behind ammonium sulfate precipitation for protein purification?
Answer: High salt concentrations remove the hydration shell from proteins reducing their solubility until they precipitate (salting out)
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).
In a Western blot procedure, what is the function of the blocking step (e.g., 5% milk or BSA)?
Answer: To saturate non-specific protein-binding sites on the membrane preventing antibody from binding non-specifically to the membrane background
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.
What does 'affinity chromatography' exploit to purify a specific protein from a complex mixture?
Answer: The specific, reversible, non-covalent interaction between the target protein and an immobilized ligand
Affinity chromatography uses a column containing an immobilized ligand that specifically binds the target protein; after washing away non-binding proteins, the target is eluted by disrupting the interaction.