Which reagent is used to differentiate LD isoenzymes on gels?

• A. Nitrotetrazolium blue
• B. Silver nitrate
• C. Coomassie Brilliant Blue
• D. Sudan

Answer: (A)

Differentiating LD isoenzymes on gels relies on staining for enzyme activity after the electrophoretic separation. The trick is a staining step that reports LDH activity directly. Nitrotetrazolium blue (NBT) is used in combination with NADH (and lactate as substrate) so that where LDH is active, the enzyme converts lactate to pyruvate, reducing NAD+ to NADH. The NADH then reduces NBT via the staining system, producing a blue (formazan) precipitate exactly at the locations of the LDH isoenzymes. This reveals distinct bands corresponding to the different LDH forms.

The other reagents don’t reflect LDH activity in this context. Silver nitrate is used for general protein staining, not for enzyme activity; Coomassie Brilliant Blue stains total protein rather than activity; and Sudan dyes stain lipids.