Protein Isolation and Quantification Flashcards
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What is the purpose of the 'stacking gel' in a discontinuous SDS-PAGE system (Laemmli system)?
Answer: The stacking gel (low % acrylamide, low pH) concentrates all proteins into a thin band before they enter the resolving gel
The stacking gel (3-5% acrylamide, pH 6.8) uses the Laemmli discontinuous buffer system to concentrate proteins into a sharp band at the gel interface before they enter the resolving gel, improving band sharpness.
In a bicinchoninic acid (BCA) protein assay, what reaction produces the color change used for quantification?
Answer: Protein reduces Cu2+ to Cu1+ in alkaline conditions; BCA chelates Cu1+ to form a purple complex measured at 562 nm
The BCA assay uses two reactions: biuret reaction where protein reduces Cu2+ to Cu1+ in alkaline conditions, then BCA chelates Cu1+ ions to form an intense purple product (ฮปmax 562 nm).
What technique separates proteins based on their isoelectric point (pI)?
Answer: Isoelectric focusing (IEF)
Isoelectric focusing applies a pH gradient to a gel or capillary; proteins migrate until they reach the pH region equal to their pI, where they carry no net charge and stop migrating.
In protein purification, what does 'specific activity' measure, and why does it increase during successful purification?
Answer: Specific activity measures enzyme activity per unit total protein (units/mg); it increases during purification as contaminant proteins are removed enriching the target enzyme
Specific activity = total activity / total protein; as purification removes non-target proteins, total protein decreases while target enzyme activity is maintained, increasing specific activity.
What is the principle of size-exclusion chromatography (SEC, or gel filtration) for protein separation?
Answer: Small proteins enter pores in the gel matrix and elute last; large proteins excluded from pores elute first in order of decreasing size
SEC separates proteins by size: large proteins excluded from pores elute first, while small proteins enter pores, take a longer path, and elute later.
What is the purpose of the 'transfer' step in a Western blot procedure?
Answer: To electrophoretically transfer proteins from the SDS-PAGE gel onto a membrane (nitrocellulose or PVDF) for antibody-based detection
Transfer moves the separated proteins from inside the polyacrylamide gel onto a membrane surface, making them accessible for antibody binding in subsequent immunodetection steps.