Aladdin ® 488 (6) -xSE (Aladdin ® 488 (6) -x succinimidyl ester)

aladdin® SE (or NHS ester)是我司生产的具有氨基反应活性的一类荧光染料。该类染料的SE基团可以与氨基基团反应生产稳定的酰胺键。相比于市面上其他同类染料，aladdin®是稳定性更强、水溶性更好、荧光强度更优的新一代荧光染料。 我司还提供小批量抗体标记试剂盒aladdin® Antibody Labeling Kits，可在30 min内标记5-100 μg抗体，而无需纯化步骤，简单方便。 产品参数： Absmax/Em(nm)：491/512；A280/Amax or Cf (protein)：0.1 ；Extinction coefficient(ε)：76,000；Optimal DOL(IgG)：7-9 ；MW：947.1 使用方法（以标记 IgG 抗体为例） 1. 实验材料 （1）IgG：IgG 不可含有能与染料反应的胺类化学物质，如氨基酸、Tris、BSA、明胶等。如果IgG 中含有此类化学物质，应用pH~7.4 的PBS 缓冲液预先透析处理。叠氮类化合物的存在不会影响标记反应。 （2）无水DMSO （3）NaHCO3 （4）葡聚糖凝胶G-25 透析柱 （5）PBS 缓冲液（pH~7.4） （6）NaN3 （7）BSA 2. 标记方法和步骤 （1）准备标记抗体 用0.1 M 的NaHCO3 溶液（pH~8.3）稀释抗体，使抗体终浓度为2.5 mg/mL。如果产品预先用磷酸盐缓冲液稀释， 如PBS 缓冲液（不含胺基类化合物），那么可以直接在该缓冲液中加入约1/10 体积1M 的NaHCO3 母液，使NaHCO3 终浓度为0.1 M。 注：蛋白浓度为2.5 mg/mL 时，标记效率大概为35%，蛋白浓度低于2.5 mg/mL 也可用于标记，但标记效率会降低。当蛋白浓度高于5 mg/mL 时，标记效率可能更高。由于缓冲液和蛋白纯度存在差异性，因而更精确的标记效率由实操条件决定。如果蛋白浓度过低，可以通过超滤法进行浓缩。 （2）准备染料储存液 室温预热一管1 μmole 的YF®SE，在管中加入0.1 mL 的无水DMSO，充分涡旋溶解染料，配制浓度为10 mM 的染料储存液。如果使用更微量的蛋白进行标记反应，那么染料需要稀释至更低浓度。 注：a. 剩余的染料储存液应于-20℃低温存放，以备后续使用。如果使用无水DMSO 配制染料储存液，那么染料至少可以保存一个月。 b. 染料也可以用去离子水配制，但是由于染料在水中会缓慢水解，所以水配制的储液最好现配现用。 （3）标记反应步骤 a. 搅拌或涡旋混匀蛋白溶液，逐步滴加15-25 μL 的染料储存液（10 mM），使染料/蛋白的摩尔比在9: 1 至15: 1 的范围内。YF®SE 标记IgG 抗体的DOL（结合于每个蛋白分子上的染料数量）范围请参考上表。 b. 在室温下搅拌反应1 h，微量标记时也可在摇床上振荡孵育1 h。 注：在进行结合反应的同时，进行步骤2(4)，平衡葡聚糖凝胶G-25 透析柱。 （4）从反应液中分离标记蛋白 a. 用PBS 缓冲液（pH~7.4）平衡葡聚糖凝胶G-25 透析柱（10 mm×300 mm）。 b. 将步骤3(b)反应溶液加入柱子，并用1×PBS 缓冲液洗脱。首先洗脱出来的着色带是染料-蛋白结合物。 注：a. 对于小规模的标记反应，为了避免过度稀释产物，可以使用超滤装置去除结合物中的自由染料。 b. 当结合反应完成后，如不及时分离染料-蛋白结合物， 可以加入50 μL 1M 赖氨酸终止反应。多数情况下，不需要此操作，因为剩余的未反应的染料在反应最后已经被充分水解。 3. 确定 DOL （1）蛋白浓度的确定 抗体浓度可通过以下公式计算： C(mg/mL)={[A280 -(Amax × Cf)]/1.4} × 稀释因子； a. C 是指实验收集的抗体浓度； b. 稀释因子是指在光度测量时的稀释倍数； c. A280 和Amax 分别是指在280 nm 处的吸光度以及在吸收波长处的吸光度； d. Cf 是校正因子，YF®SE 染料的Cf 值请参考上面表格； 注：过柱洗脱的蛋白溶液直接用于吸光度检测可能浓度过大， 因此需要稀释到大约0.1 mg/mL。稀释倍数（即稀释因子） 需要从起初抗体数量（比如5 mg）以及蛋白液洗脱的总体积来进行预估。 （2）DOL 的估算 DOL 通过下式计算： DOL = (Amax × Mwt × 稀释因子)/(ε × C) a. Amax，稀释因子，C 值在3(1)中已经明确； b. Mwt 是指IgG 的分子量(150,000)； c. ε 是YF®SE 的摩尔吸光系数，参考第一页表格； d. 标记YF®SE 的IgG 抗体最适DOL 值，DOL 值会波动，但也能得到很好的实验效果。 Product introduction：Aladdin ® SE (or NHS ester) is a kind of fluorescent dye with amino reaction activity produced by our company.SE groups of these dyes can react with amino groups to produce stable amide bonds. Compared with other similar dyes on the market, Aladdin ® It is a new generation of fluorescent dyes with stronger stability, better water solubility and better fluorescence intensity. Our company also provides a small batch of antibody labeling kit Aladdin ® Antibody labeling kits ， which can label 5-100 in 30 min μ G antibody without purification steps, which is simple and convenient. Product parameters: Product parameters：Absmax/Em(nm)：491/512；A280/Amax or Cf (protein)：0.1 ；Extinction coefficient(ε)：76,000；Optimal DOL(IgG)：7-9 ；MW：947.1 Usage method (using labeled IgG antibodies as an example) 1. Experimental materials (1) IgG: IgG must not contain amine chemicals that can react with dyes, such as amino acids, Tris, BSA, gelatin, etc. If IgG contains such chemicals, PBS buffer with pH~7.4 should be used for pre dialysis treatment. The presence of azide compounds does not affect the labeling reaction. (2) Anhydrous DMSO (3) NaHCO3 (4) Sephadex gel G-25 dialysis column (5) PBS buffer (pH~7.4) (6) NaN3 (7) BSA 2. Marking methods and steps (1) Prepare to label antibodies Dilute the antibody with 0.1 M NaHCO3 solution (pH~8.3) to a final concentration of 2.5 mg/mL. If the product is pre diluted with phosphate buffer, such as PBS buffer (without amino compounds), approximately 1/10 volume of 1M NaHCO3 mother liquor can be directly added to the buffer to achieve a final NaHCO3 concentration of 0.1 M. Note: When the protein concentration is 2.5 mg/mL, the labeling efficiency is approximately 35%. Protein concentrations below 2.5 mg/mL can also be used for labeling, but the labeling efficiency will decrease. When the protein concentration is higher than 5 mg/mL, the labeling efficiency may be higher. Due to differences in buffer and protein purity, more precise labeling efficiency is determined by practical operating conditions. If the protein concentration is too low, it can be concentrated by ultrafiltration. (2) Prepare dye storage solution Preheat one tube at room temperature μ YF of Mole ® SE, add 0.1 mL of anhydrous DMSO to the tube, thoroughly vortex dissolve the dye, and prepare a dye storage solution with a concentration of 10 mM. If a trace amount of protein is used for labeling reactions, the dye needs to be diluted to a lower concentration. Note: a The remaining dye storage solution should be stored at a low temperature of -20 ℃ for future use. If anhydrous DMSO is used to prepare dye storage solution, the dye can be stored for at least one month. b. Dyes can also be prepared with deionized water, but due to the slow hydrolysis of dyes in water, it is best to prepare water based storage solutions for immediate use. (3) Mark reaction steps a. Stir or vortex the protein solution, gradually adding 15-25 drops μ L dye storage solution (10 mM), with a molar ratio of dye/protein in the range of 9:1 to 15:1. YF ® Please refer to the table above for the range of DOL (number of dyes bound to each protein molecule) for SE labeled IgG antibodies. b. Stir the reaction at room temperature for 1 hour, and for trace labeling, shake and incubate on a shaker for 1 hour. Note: At the same time of the binding reaction, proceed to step 2 (4) to balance the dextran gel G-25 dialysis column. (4) Isolation of marker proteins from reaction solution a. PBS buffer (pH~7.4) was used to balance the dextran gel G-25 dialysis column (10 mm × 300 mm). b. Add the reaction solution from step 3 (b) to the column and elute with 1 x PBS buffer. The first washed out chromophore is a dye protein complex. Note: a For small-scale labeling reactions, in order to avoid excessive dilution of the product, ultrafiltration devices can be used to remove free dyes from the complex. b. After the binding reaction is completed, if the dye protein complex is not separated in time, 50 can be added μ Terminate the reaction with L 1M lysine. In most cases, this operation is not necessary because the remaining unreacted dyes have been fully hydrolyzed at the end of the reaction. 3. Determine DOL (1) Determination of protein concentration The antibody concentration can be calculated using the following formula: C (mg/mL)={[A280- (Amax x x Cf)]/1.4} x dilution factor; a. C refers to the concentration of antibodies collected in the experiment; b. Dilution factor refers to the dilution factor used in photometric measurements; c. A280 and Amax refer to the absorbance at 280 nm and the absorbance at the absorption wavelength, respectively; d. Cf is the correction factor, YF ® Please refer to the table above for the Cf value of SE dyes; Note: The protein solution eluted through the column may have a high concentration when used directly for absorbance detection, so it needs to be diluted to approximately 0.1 mg/mL. The dilution factor (i.e. dilution factor) needs to be estimated based on the initial number of antibodies (e.g. 5 mg) and the total volume of protein elution. (2) Estimation of DOL DOL is calculated using the following equation: DOL=(Amax x x Mwt x Dilution Factor)/（ ε X C) a. Amax, dilution factor, C value has been clearly defined in 3 (1); b. Mwt refers to the molecular weight of IgG (150000); C. c ε It's YF ® The molar absorption coefficient of SE, refer to the table on the first page; d. Mark YF ® The optimal DOL value for SE IgG antibodies can be found in the table on the first page. Although DOL values may fluctuate, good experimental results can also be achieved.

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