5(6)-Carboxyfluorescein (5-(and-6)-Carboxyfluorescein mixed isomers) contains a carboxylic acid that can be used to react with primary amines via carbodiimide activation of the carboxylic acid.

Instructions
1. Labeling process:
1) 5(6)-Carboxyfluorescein reacts with amino-containing biomolecules (such as proteins or peptides) to form a stable covalent bond. The commonly used reaction conditions are neutral or slightly alkaline pH environment.
2) The reaction needs to be carried out in an appropriate buffer, usually phosphate buffered saline (PBS) or other biocompatible buffers. The reaction time is adjusted according to the concentration of the target molecule and the labeling requirements.
2. Post-labeling treatment:
1) After the reaction is completed, dialysis or centrifugation is used to remove unreacted dye to ensure the purity of the labeled molecule.
2) The labeled protein or peptide can be used in a variety of downstream applications, such as fluorescence microscopy, flow cytometry analysis, fluorescence quantitative PCR, etc.
3. pH-sensitive properties:
5(6)-Carboxyfluorescein is pH-dependent, and its fluorescence intensity changes under different pH conditions. This makes it an ideal choice for studying intracellular pH changes (such as acidic areas in tumor microenvironments).