5-ROX (5-Carboxy-X-rhodamine), a rhodamine dye, exhibits strong fluorescence in aqueous buffer with an excitation wavelength (λexit) of 580 nm (ε = 3.6×10^4/(M·cm)) and an emission wavelength (λemit) of 604 nm (ϕ = 0.94).

I. Nucleic acid or protein labeling
1. Material preparation:
1) 5-ROX dye (usually in the form of NHS ester or succinimidyl ester).
2) Nucleic acid or protein sample.
3) Buffer (such as 0.1 M NaHCO₃, pH 8.5).
4) Organic solvent (such as DMSO or DMF, used to dissolve the dye).
2. Labeling steps:
1) Dissolve the dye: Dissolve 5-ROX dye in anhydrous DMSO or DMF, usually at a concentration of 10 mM.
Note: The dye solution should be kept away from light and used as soon as possible.
2) Prepare the labeling reaction: Add 5-ROX solution to the nucleic acid or protein sample, usually at a molar ratio of 10:1 (dye: target molecule); add an appropriate amount of buffer (such as 0.1 M NaHCO₃, pH 8.5) to the reaction system to ensure that the reaction environment is suitable for the formation of amide bonds.
3) Incubation: Incubate at room temperature for 1–2 hours (protein labeling) or 2–4 hours (nucleic acid labeling), avoiding strong light exposure.
4) Purification: Use ultrafiltration centrifuge tubes, gel filtration columns or HPLC to purify unbound dye molecules.
5) Storage: Dissolve the labeled product in an appropriate buffer (such as TE buffer) and store at -20°C away from light.
II. Real-time PCR (qPCR) application
1. Material preparation:
1) 5-ROX fluorescent dye.
2) Real-time PCR reaction mixture (including template, primers, probe and polymerase).
3) Appropriate fluorescence detection equipment (such as qPCR instrument with orange-red light channel).
2. Usage steps:
1) Add 5-ROX: Add an appropriate amount of 5-ROX according to the equipment requirements (usually a final concentration of 50–100 nM per 50 μL reaction system).
2) Mix well: Gently mix the reaction solution to avoid bubbles.
3) Run PCR: Detect fluorescence signals within the wavelength range supported by the device (excitation ~580 nm, emission ~600 nm).
Notes:
1) Avoid light: 5-ROX is light-sensitive and should be kept away from light as much as possible during operation and storage. It is recommended to store it at 4°C for short term and -20°C for long term.
2) Solvent selection: Use anhydrous DMSO or DMF to ensure the stability of the dye.
3) Optimize labeling efficiency: The pH and dye concentration of the sample should be adjusted according to the characteristics of the target molecule to optimize the labeling efficiency.
4) Safety: Avoid skin contact and inhalation, and wear gloves and goggles during operation.