You are purifying a protein by column chromatography. What indicates that you have done enough washes and that you can begin eluting?

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We are considering a question about protein purification by column chromatography and deciding when to start eluting. The key concept is monitoring the wash fractions to ensure that residual contaminants are removed and that most of the target protein has not yet eluted. The options describe different observable properties of the wash fractions.
Option 1: 'The wash fractions have become very viscous' - Viscosity is not a standard or reliable indicator used to decide when to switch from washing to eluting. Protein solutions can vary in viscosity due to buffers or additives, but a rise in viscosity does not reliably indicate complete removal of contaminants or readiness for elution. This makes this option misleading as a criterion.
Option 2: 'The wash fractions have very low A280' - Absorbance at 280 nm (A280) is commonly used to monitor protein content because aromatic amino acids absorb UV light at 280 nm. When wash fractions show very low A280, it suggests that most protein (including the target protein) is being removed during washing, and it may be appropriate to begin elution once the baseline is reached and A280 is minimal, indicating wash steps have cleared loosely bound contaminants. This aligns with standard practice of using a sharp drop in A280 to decide that you have adequately washed.
Option 3: 'The wash fractions are colorless' - Colorlessness of wash fractions is not a standard, quantitative criterion for deciding when to elute. Color can vary due to buffer components or impurities, and a lack of color does not reliably indicate removal of contaminants or readiness for elution.
Option 4: 'The wash fractions show precipitation' - Precipitation in wash fractions indicates protein instability or incompatibility and is generally a sign of problems in purification, not a confirmation that washing is complete or that elution should begin. This would typically prompt troubleshooting rather than proceeding to elution.
Option 5: 'The wash fractions have very low A380' - Absorbance at 380 nm is less commonly used for protein monitoring; A380 is sometimes related to dye binding or other assays, not a standard indicator of successful washing in typical protein purification by chromatography. Therefore, relying on A380 is not a correct criterion for deciding to elute.
In summary, among the given options, the most appropriate criterion that aligns with standard purification practices is that the wash fractions have very low A280, indicating that protein from the wash is minimal and you can consider moving to elution once you’ve cleared the wash step. The other options describe observations that are not reliable indicators of when to begin elution and can mislead the decision-making process.

CHEM108_WI25_A00 Final Exam

You are purifying a protein by column chromatography. What indicates that you have done enough washes and that you can begin eluting?

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