Deep Red Fluorescent Rapid Labeling Kit (CY5）
Deep Red Fluorescent Rapid Labelling Kit (CY5) enables red fluorescent labelling of proteins. Maximum excitation/ emission wavelengths: 649/667 nm. Based on NHS ester chemistry, the NHS ester-activated fluorescent dye reacts with primary amines in the antibody/protein to be labelled at pH 7-9, forming stable amide bonds to achieve conjugation. Typically, one IgG molecule can bind 2–8 molecules of CY5. The entire experiment can be completed within 2 hours.
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Storage :
4°C 1 year
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Description & Advantages
Fluorescent labelling services involve covalently binding or physically adsorbing fluorescent substances onto specific functional groups of target molecules. Leveraging fluorescent properties, this technique enables qualitative, localisation and quantitative analysis of labelled subjects. Offering advantages such as absence of radioactive contamination, straightforward operation and ease of observation, its applications have penetrated numerous fields including pharmacology, physiology, environmental science and information science. It also finds extensive use in protein functional studies and drug screening.
Deep Red Fluorescent Rapid Labelling Kit (CY5) enables red fluorescent labelling of proteins. Maximum excitation/ emission wavelengths: 649/667 nm. Based on NHS ester chemistry, the NHS ester-activated fluorescent dye reacts with primary amines in the antibody/protein to be labelled at pH 7-9, forming stable amide bonds to achieve conjugation. Typically, one IgG molecule can bind 2–8 molecules of CY5. The entire experiment can be completed within 2 hours.
Protocol
3. Kit Usage Instructions
3.1 Sample Preparation
Prepare antibodies/proteins for labelling
1) Buffer pH requirement: pH 6.5–8.5.
2) Buffer component requirements: Ideal buffers: PBS; HEPES; potassium salts; sodium salts.
3) Restricted components: Tris ≤ 50 mM / 0.6%; BSA ≤ 0.1%; Glycerol ≤ 10%.
4) Prohibited components: Arginine; Glutathione; DTT.
Note: Should the buffer fail to meet requirements, sample dialysis in PBS is necessary. Microdialysis cups may be employed for small sample volumes.
5) Protein concentration must exceed 1 mg/mL, with 2 mg/mL being optimal. If concentration is too low, concentrate using an ultrafiltration tube.
3.2 Preparation of Dye Stock Solution
Dissolve lyophilised powder using the dye dissolving solution. Add the dissolving solution to the dye and prepare for use.
1) Kit specification (50–200 μg): Add 25 μl;
2) Kit specification (200μg–1mg): Add 110μl.
3.3 Labelling
Antibody (protein) quantity: Add the dissolved dye to the antibody/protein to be labelled* at a volume ratio of 1:10 (e.g., add 5μl dye per 50μg antibody/protein). Mix thoroughly, place in a light-protected bag, and incubate on a shaker or rotator for 30 minutes to 1 hour. ;
*Minor flocculation may occur during addition; gently pipette several times and mix thoroughly; flocculation will dissipate.
*Rotation for 30 minutes achieves 90% reaction efficiency; 3 hours yields >98% efficiency. To enhance efficiency, extend reaction time or incubate overnight at 4°C.
3.4 Purification
3.4.1 Purification Column Preparation
Remove the sealing foil. Centrifuge at 1000 g for 2 minutes. Discard the supernatant from the outer tube. Transfer the inner tube of the purification column to a 1.5 ml collection tube and set aside.
3.4.2 Collection
After brief centrifugation, transfer the entire contents to the inner tube of the purification column. Allow to stand for 1 minute, then centrifuge at 1000 g for 2 minutes. Collect the labelled product and discard the inner tube of the purification column.
Storage
4°C 1 year
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