Tracking a protein during purification: What is the most reliable way to locate your target enzyme in fractions collected from each step?

Introduction / Context:
Purifying an enzyme requires a reliable readout to identify which fractions contain active protein. Activity assays directly report functional presence, making them superior to non-specific signals.

Given Data / Assumptions:

• Target is an ATP-synthesizing enzyme (example used here).
• Fractions from steps such as precipitation or chromatography are available.
• Access to a simple, quantitative activity assay.

Concept / Approach:
While UV at 280 nm reveals total protein, it cannot distinguish the target from contaminants. Refractive index, conductivity, or broad mass spectrometry screens are non-specific or impractical at every step. A tailored activity assay is the gold standard for tracking the target and calculating specific activity and yield.

Step-by-Step Solution:
Establish an assay that measures ATP formation per unit time.Test each fraction; identify those with the highest specific activity.Pool active fractions and proceed to the next step.

Verification / Alternative check:
Correlate activity peaks with SDS-PAGE bands; specific activity should rise as purification advances.

Why Other Options Are Wrong:

• UV 280 nm: Non-specific; many proteins absorb.
• Refractive index/conductivity: Reflect buffer composition, not enzyme identity.
• Mass spectrometry: Powerful but not routine for every fraction during process development.

Common Pitfalls:
Relying solely on A280 can mislead pooling decisions; always confirm with activity.

Final Answer:
Measure the rate of ATP synthesis (an activity assay specific to the target).