DNP-X, SE (6-(2,4-Dinitrophenyl)aminohexanoic acid, succinimidyl ester) is an amine-reactive building block recognized by anti-DNP antibodies and serves as an excellent amine-reactive FRET quencher when paired with Trp or Tyr.

Instructions
1. Solvent selection
DNP-X, SE is easily soluble in anhydrous DMSO or DMF. Make sure the solvent is anhydrous to avoid hydrolysis of succinimide esters.
2. Sample preparation
Target molecule: must contain free amine groups (such as lysine side chains or N-terminal amine groups of proteins).
Buffer selection: It is recommended to use an amine-free buffer (such as carbonate or phosphate buffer) with a pH of about 8.3. Avoid Tris or other amine-containing buffers to avoid interference with the reaction.
3. Coupling reaction
a. Prepare a DMSO solution of DNP-X, SE (usually 10 mM concentration).
b. Add the target molecule to the buffer, and then add the DNP-X, SE solution dropwise (use the reagent in a 10–20-fold excess of the molar amount of the target molecule).
c. React at room temperature or slightly below room temperature (4°C) for 30 minutes to 2 hours, away from light and high temperature.
d. Adjust the pH of the reaction between 8–9 to maintain the activity of the succinimidyl ester.
4. Reaction termination and purification
a. Adjust the pH of the reaction solution to neutral (7.0) with a buffer.
b. Remove unreacted DNP-X, SE and byproducts by one of the following purification methods:
Dialysis: Suitable for labeling large molecules (such as proteins).
Gel filtration columns (such as Sephadex G-25).
HPLC: Suitable for high-precision purification.
5. Verify the labeling
UV-Vis spectrophotometry: The absorption peak of DNP-X is usually in the range of 360–400 nm.
FRET detection: Detect the quenching effect of energy transfer when paired with Trp or Tyr as a donor.
Notes
a. Storage conditions: DNP-X, SE should be stored at -20°C, away from light and moisture, and can be stored under inert gas (such as nitrogen) when necessary.
b. Avoid cross-reactions: Ensure that the target molecule sample does not contain other free amine groups or reactive groups that interfere with labeling.
c. Optimize conditions: Adjust reagent concentration, reaction time and temperature according to the characteristics of the target molecule to maximize labeling efficiency.