Choosing separation strategies in protein purification: On which physicochemical properties can proteins be separated?

Introduction / Context:
Successful purification usually combines orthogonal methods that exploit different properties of proteins. Recognizing available handles streamlines the design of a robust workflow.

Given Data / Assumptions:

• Proteins vary in charge (pI), size, hydrophobicity, and solubility.
• Common techniques include ion exchange, salting-out, hydrophobic interaction, and size-exclusion chromatography.
• Target protein is stable under chosen conditions.

Concept / Approach:
Charge differences resolve proteins on ion exchangers. Solubility manipulations (for example, ammonium sulfate precipitation) concentrate proteins and remove contaminants. Size differences are exploited by gel filtration (SEC), which sieves molecules by hydrodynamic radius.

Step-by-Step Solution:
Screen conditions to find where target binds/precipitates appropriately.Sequence columns to maximize resolution with minimal denaturation.Monitor fractions by activity and SDS-PAGE.

Verification / Alternative check:
Orthogonality test: if two methods rank proteins differently, they complement each other and improve purity.

Why Other Options Are Wrong:

• Only hydrophobicity: Overlooks many effective and widely used strategies.

Common Pitfalls:
Using methods that are not orthogonal, leading to marginal improvements; ignoring pH/pI alignment for ion exchange.

Final Answer:
All of these.