Question

Which technique separates proteins based on isoelectric point?

• A. SDS-PAGE
• B. Isoelectric focusing
• C. Gel filtration
• D. Affinity chromatography

Hint:

The name of the technique often reveals its exact mechanism. "Isoelectric" directly points to the "isoelectric point" (pI). Remember that 2D gel electrophoresis utilizes IEF as the first dimension (separation by pI) and SDS-PAGE as the second dimension (separation by size).

Answer 1

The Correct Option is B

Step 1: Understanding the Concept:
Proteins are complex macromolecules that can be separated using various biochemical techniques.
These techniques exploit distinct physical and chemical properties of the proteins, such as their molecular size, net charge, or specific binding affinities.

Step 2: Detailed Explanation:
Let's analyze the primary separation principle of each listed technique.
- Isoelectric focusing (IEF): This is an electrophoretic technique where proteins are driven through a gel containing a continuous pH gradient under an electric field.
A protein will migrate through the gel until it reaches the specific pH region that is exactly equal to its isoelectric point (pI).
At this precise pH, the protein's net charge becomes zero, and it stops migrating, meaning it separates purely based on its pI.
- SDS-PAGE: This technique uses Sodium Dodecyl Sulfate to denature proteins and coat them with a uniform negative charge.
Consequently, the proteins separate primarily based on their molecular weight (size) as they migrate toward the positive electrode.
- Gel filtration (Size-exclusion chromatography): This column chromatography method separates molecules based purely on their physical size and shape as they pass through porous beads.
- Affinity chromatography: This method separates proteins based on highly specific biological interactions, such as an enzyme binding to its substrate or an antibody binding to its antigen.

Step 3: Final Answer:
Isoelectric focusing is the only technique listed that isolates proteins based on their isoelectric point.
Therefore, option (B) is the correct answer.