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.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.

H2O: 25 mg/mL (39.63 mM), Sonication is recommended.