Red Fluorescent Rapid Labeling Kit (AF555） 

红色荧光快速标记试剂盒（AF555）

荧光标记服务指将荧光物质共价结合或物理吸附在所要研究分子的某个基团上，借助荧光特性对被标记对象进行定性、定位以及定量分析，具有无放射物污染、操作简便、容易观察等优点，其应用已深入到药物学、生理学、环境科学、信息科学等诸多领域，在蛋白的功能研究、药物筛选等领域也具有广泛的应用。 红色荧光快速标记试剂盒（AF555）可对蛋白进行红色荧光标记，最大激发/发射波长：555/565 nm，基于NHS酯反应化学，NHS酯活化的荧光染料和待标记抗体/蛋白在pH7-9溶液中与伯胺反应，形成稳定的酰胺键，从而实现与抗体/蛋白的偶联。一般一 个 IgG 分子可结合 2-8 个分子的 AF555。整体实验可在2小时内完成。

4°C 1年

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