Two-dimensional electrophoresis – What property is used in the first-dimension separation before SDS-PAGE in standard 2D gels?

Introduction / Context:
Two-dimensional (2D) gel electrophoresis resolves complex protein mixtures by separating proteins according to two independent properties. The first dimension uses isoelectric focusing (IEF), which separates proteins by their isoelectric point (pI). The second dimension applies SDS-PAGE to separate proteins by molecular mass. This orthogonality greatly enhances resolving power for proteomics and differential expression studies.

Given Data / Assumptions:

• Proteins carry different net charges at different pH values.
• In IEF, proteins migrate in a stabilized pH gradient until net charge is zero (at pI).
• SDS-PAGE then masks charge with SDS, making separation primarily size-based.

Concept / Approach:
Because pI and molecular mass are largely independent, combining IEF with SDS-PAGE spreads proteins across a 2D map: horizontal location reflects pI, vertical position reflects size. Staining (e.g., Coomassie, silver) or fluorescent dyes visualize the spots. Spots can be excised for mass spectrometric identification.

Step-by-Step Solution:

Verification / Alternative check:
Proteins with identical mass but different pI (e.g., isoforms or PTM variants) resolve horizontally in IEF, confirming that the first dimension is based on isoelectric point rather than size or solubility.

Why Other Options Are Wrong:

• Molecular mass: used in the second dimension, not the first.
• Solubility, folding, hydrophobicity: not the primary variables exploited in standard 2D electrophoresis.

Common Pitfalls:
Confusing IEF-first with blue native PAGE workflows; in classic 2D gels, the first dimension is always IEF by pI.

Final Answer:
Isoelectric point (pI) via isoelectric focusing)