NBD-Cl (NBD chloride) is a nonfluorescent compound that becomes highly fluorescent after reaction with amino or thiol groups.

1. Protein labeling
Experimental steps:
1. Prepare NBD-Cl solution: Dissolve NBD-Cl in an appropriate organic solvent, such as dimethylsulfide (DMSO), and prepare a stock solution with a concentration of usually 1-10 mM.
2. Reaction with protein: Mix the NBD-Cl solution with a protein sample containing free thiol or amino groups. The reaction is usually carried out at room temperature or slightly alkaline to facilitate the reaction with the thiol group.
3. Incubation: The reaction incubation time is usually 30 minutes to 1 hour to ensure the reaction is complete.
4. Purification: After the reaction is completed, the unreacted NBD-Cl can be removed by dialysis or filtration to ensure that only the labeled protein is left.
5. Fluorescence measurement: Fluorescence emission is measured using a fluorescence spectrophotometer, usually with excitation wavelength of 460 nm and emission wavelength of approximately 530 nm to confirm the labeling efficiency.
2. Quantification of thiol groups
Experimental steps:
1. Prepare NBD-Cl solution: Dissolve NBD-Cl in DMSO or other appropriate solvent to prepare a stock solution.
2. Sample Preparation: Add NBD-Cl to a biological sample or buffer containing free thiol. The concentration of NBD-Cl should be optimized to ensure complete reaction with the thiol group.
3. Incubation: The reaction incubation time is usually 10-30 minutes, allowing the thiol to react with NBD-Cl.
4. Fluorescence measurement: Use a fluorescence meter to measure the emission spectrum, the emission wavelength is usually 530 nm and the excitation wavelength is 460 nm. The fluorescence intensity is proportional to the number of thiol groups in the sample.
III. Amino detection
Experimental steps:
1. Prepare NBD-Cl solution: Dissolve NBD-Cl in DMSO or other appropriate solvent to prepare a stock solution.
2. Sample preparation: Add NBD-Cl solution to a sample containing free amino groups (such as small molecules or peptides).
3. Incubation: The reaction is usually carried out at room temperature and the incubation time is 15-30 minutes.
4. Fluorescence measurement: Use a fluorescence meter to measure the emission spectrum, which is usually 530 nm and excitation wavelength is 460 nm, to detect the labeling of amino groups.