DNP-X acid (6-((2,4-Dinitrophenyl)amino)hexanoic acid) 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. Add appropriate amount of DNP-X acid and Trp or Tyr to the reaction system.
2. Select appropriate reaction conditions and use chemical coupling reagents such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) to promote covalent bonding between amine and carboxyl groups.
3. After the reaction is completed, dialysis, chromatography and other methods can be used to remove unreacted substances to obtain coupling products.
3. FRET system establishment and use of DNP group as a quencher, pair it with a fluorescent marker with suitable excitation and emission wavelengths (such as GFP or other fluorescent dyes) to establish a FRET system:
1) Combine the DNP-X acid coupling product with a fluorescent molecule (such as green fluorescent protein or dye).
2) By exciting the fluorescent molecule, the fluorescence change caused by the quenching effect of the DNP group can be detected under the FRET mechanism.
4. Binding with anti-DNP antibody:
1) When the probe binds to the anti-DNP antibody, the change in FRET signal (such as the change in fluorescence intensity) can be used to quantify the antibody or detect antibody binding.
2) The increase or decrease in FRET signal reflects the binding of the antibody, which is then used for antibody quantification and detection.
5. Usage and dosage of DNP-X acid
1. Preparation of DNP-X acid solution: DNP-X acid is usually prepared into a solution of appropriate concentration, with a common concentration range of 1-10 mM (the specific concentration is optimized according to experimental needs).
2. Dosage of coupling reaction: Usually, about 1-2 equal molar ratios of DNP-X acid and Trp or Tyr are added per reaction (depending on the concentration of the target molecule and the reaction efficiency). For example, if the concentration of the target molecule is 1 mM, 1-2 mM DNP-X acid can be added.
3. Coupling reaction time: The coupling reaction generally takes 1-2 hours. The temperature can be room temperature or slightly elevated (such as 37°C), but avoid excessively high temperatures that may cause inactivation of the reaction.