CY3 (Sulfo-Cyanine3) is an orange-fluorescent label for nucleic acids and proteins (λex=554, λem=568).

1. Protein labeling
Experimental steps:
1. Prepare CY3 dye solution: Dissolve CY3 in an appropriate solvent (such as DMSO or PBS), at a concentration of usually between 1-10 μM.
2. Reaction with antibodies or proteins: Incubate CY3 dye (10 mg/ml) with the target protein or antibody. Incubate normally at room temperature for 30-60 minutes to ensure the dye binds to the target molecule.
3. Purification: Use dialysis or filtration to remove unbound dyes to ensure that only labeled proteins are present in the sample.
4. Fluorescence measurement: Use a fluorescence microscope or spectrophotometer to detect labeled proteins by selecting the appropriate excitation wavelength (λex = 554 nm) and emission wavelength (λem = 568 nm).
2. Nucleic acid labeling
Experimental steps:
1. Prepare CY3-labeled probes or primers: Synthesize probes or primers with CY3-labeled, usually using conventional labeling chemistry.
2. Hybridization: Under appropriate reaction conditions, mix the CY3-labeled probe with the target nucleic acid (such as DNA or RNA) and perform a hybridization reaction.
3. Fluorescence imaging: Use a fluorescence microscope or real-time PCR system to select the appropriate wavelength to detect CY3-labeled nucleic acid signals.
3. Double marking experiment:
Experimental steps:
1. Label multiple targets: Use CY3 and other fluorescent markers (such as FITC, Cy5, etc.) to label different target molecules separately.
2. Confocal imaging: Observe multiple labeled samples through confocal microscope or multi-channel microscope to perform spatial positioning of target molecules.
Notes:
1. Photostability: CY3 dye may experience photobleaching under high-intensity light, so it is necessary to optimize the exposure time and light source intensity of fluorescence imaging experiments.
2. Solubility: CY3 has good solubility, but it is still necessary to avoid excessive concentration of dye solutions when used to prevent non-specific binding.
3. Cell permeability: CY3 has good cell permeability, but in some cases, it may be necessary to optimize experimental conditions, such as using penetrating reagents or performing cell membrane modification.