Which biochemical technique requires a pH gradient for separation?

• A. Limited proteolysis
• B. Isoelectric focusing
• C. SDS-PAGE
• D. Western blotting

Answer: (B)

Isoelectric focusing is the correct technique that requires a pH gradient for separation. This method is primarily used to separate proteins based on their isoelectric points (pI), which is the pH at which a protein carries no net electrical charge.

In isoelectric focusing, a gel or solution is prepared with a gradient of pH values. When a mixture of proteins is subjected to an electric field, each protein will migrate until it reaches the pH that corresponds to its pI. At this point, the protein will have no net charge and will therefore stop moving. This method effectively separates proteins with differing isoelectric points in a single dimension.

The other techniques mentioned do not utilize a pH gradient for separation. Limited proteolysis involves the partial digestion of proteins by specific enzymes but does not separate proteins based on charge. SDS-PAGE separates proteins primarily based on their size, with the use of SDS to impart a uniform negative charge irrespective of the protein's original charge. Western blotting is a technique for detecting specific proteins after separation but does not involve a separation method that utilizes pH gradients; instead, it typically follows gel electrophoresis to transfer proteins onto a membrane for detection.