Isoelectric focusing principle — Proteins are separated primarily based on what compositional/physicochemical property?

Introduction:
Isoelectric focusing (IEF) separates proteins along a fixed pH gradient until each focuses at its isoelectric point, the pH where net charge equals zero. Understanding pI connects sequence composition to separations in proteomics workflows.

Given Data / Assumptions:

• A stable pH gradient exists across the gel or capillary.
• Proteins migrate under an electric field until net charge is zero.
• pI depends on the balance of acidic and basic residues and their pKa values.

Concept / Approach:
Because migration stops when net charge is zero, proteins resolve sharply at pI values. This is complementary to SDS–PAGE (size-based) and can be combined in 2D electrophoresis (IEF in the first dimension, SDS–PAGE in the second).

Step-by-Step Solution:

Verification / Alternative check:
Mixing pI markers with samples confirms gradient integrity and focusing performance; spots align with expected pI values in 2D maps.

Why Other Options Are Wrong:

Common Pitfalls:
Assuming mobility ceases for all proteins at the same pH; overlooking ampholyte gradient stability; confusing IEF with charge-based native PAGE lacking a fixed gradient.

Final Answer:
Separation is by isoelectric point (pI), reflecting the overall balance of positive and negative residues.