Super Fluor 647

Super Fluor 647 活化酯的性质参数

活化酯分子量:1300
最大吸收波长(nm):647
摩尔吸光系数(M-1cm-1):239000 

最大发射波长(nm):665
A280nm /Amax:3%

Super Fluor 标记抗体的参考方案

1) 蛋白溶解：用0.1 M NaHCO3溶解至终浓度为2mg/mL

将1ml 去离子水加入84mgNaHCO3瓶中，配成1M 的碳酸氢钠溶液。振荡或吹打至完全溶解。该溶液pH 约8.3，2～6℃保存可用2 星期；若抗体是溶液，将抗体稀释至2mg/mL，再加入1/10 体积的上述碳酸氢钠溶液；若抗体是冻干粉，将1M 碳酸氢钠溶液用10 倍去离子水稀释成0.1M 后，用其将抗体溶成2mg/mL的溶液；

2）染料溶解：活化Super Flour SE染料固体粉末从冰箱放入干燥器中慢慢升至常温后，用无水DMSO将染料配置浓度为1 mg/mL。

3）标记：在1mL蛋白溶液中缓慢加入适量染料溶液，（蛋白:染料摩尔比=1:20～50；质量比可在以下范围内优化10～200ug Super Flour SE 每毫克IgG抗体）。同时在暗处常温缓慢搅拌1小时。也可以直接将蛋白溶液直接加入染料的固体粉末的试剂瓶中，同时在暗处常温缓慢搅拌1小时。

4）纯化：用透析法或者葡聚糖Sephadex纯化产品后，产品冷冻干燥成粉末或在0.01 M PBS/2mM叠氮化钠溶液中，-20℃避光储存。

5）贮存：标记好的蛋白贮存于2～6℃，避光。若纯化的蛋白偶联物浓度小于1mg/mL，加入BSA 或其他稳定蛋白1～10mg/ml。在2mM 叠氮钠存在的情况下，2～6 度可保存几个月。保存更长时间，则需分装后-20℃保存。避免反复冻融，避光保存。

计算标记比例F/P值

对于IgG 等大多数抗体来说，摩尔吸光系数为203000，F/P值在4～9 之间是最合适。

Super Flour 647标记蛋白F/P值计算：

1）用PBS 精确倍数稀释定量的少许纯化过的偶联蛋白，在1cm比色皿中测定280nm和650nm的光吸收；

2）计算样品中的蛋白浓度：蛋白摩尔浓度=(A280-(A650×0.03))×稀释倍数/203000

3）计算标记比例：每摩尔蛋白结合的染料摩尔数=A650×稀释倍数/(239000×蛋白摩尔浓度)

活性染料稳定性与储存

请注意：未开封的粉末在避光干燥-20℃可存放6月。其水溶液现配现用不能储存。任何溶解后的染料最好立即使用；无水的DMSO溶液-20°C保存最多2个星期。

Properties of Super Fluor 647 activated ester

Activated ester molecular weight: 1300
Maximum absorption wavelength (nm): 647
Molar absorption coefficient (M-1cm-1): 239000

Maximum emission wavelength (nm): 665
A280nm /Amax: 3%

Reference scheme for Super Fluor labeled antibodies

1) Protein dissolution: Dissolve with 0.1 M NaHCO3 to a final concentration of 2mg/mL

Add 1ml of deionized water to an 84mg NaHCO3 bottle to make a 1M sodium bicarbonate solution. Shake or pipette until completely dissolved. The pH of the solution is about 8.3, and it can be stored at 2-6°C for 2 weeks; if the antibody is a solution, dilute the antibody to 2mg/mL, then add 1/10 volume of the above sodium bicarbonate solution; if the antibody is a lyophilized powder, add 1M After the sodium bicarbonate solution is diluted with 10 times deionized water to 0.1M, use it to dissolve the antibody into a 2mg/mL solution;

2) Dye dissolution: After the activated Super Flour SE dye solid powder is slowly warmed to room temperature in a desiccator from the refrigerator, the dye preparation concentration is 1 mg/mL with anhydrous DMSO.

3) Labeling: Slowly add an appropriate amount of dye solution to 1 mL of protein solution, (protein: dye molar ratio = 1:20-50; the mass ratio can be optimized within the following range of 10-200ug Super Flour SE per mg of IgG antibody). At the same time, slowly stir for 1 hour at room temperature in the dark. It is also possible to directly add the protein solution directly to the reagent bottle of the solid powder of the dye, and at the same time, slowly stir for 1 hour at room temperature in the dark.

4) Purification: After purifying the product by dialysis or Sephadex, the product is freeze-dried into powder or stored in 0.01 M PBS/2mM sodium azide solution at -20°C and protected from light.

5) Storage: Store the labeled protein at 2～6℃, away from light. If the concentration of the purified protein conjugate is less than 1 mg/mL, add BSA or other stable proteins at 1-10 mg/ml. In the presence of 2mM sodium azide, it can be stored for several months at 2-6 degrees. For longer storage, you need to store at -20°C after packaging. Avoid repeated freezing and thawing, and store in the dark.

Calculate the mark ratio F/P value

For most antibodies such as IgG, the molar absorption coefficient is 203000, and the F/P value between 4-9 is the most appropriate.

Super Flour 647 labeled protein F/P value calculation:

1) Dilute quantitatively a few purified coupled proteins with PBS with precise multiples, and measure the light absorption at 280nm and 650nm in a 1cm cuvette;

2) Calculate the protein concentration in the sample: protein molar concentration=(A280-(A650×0.03))×dilution multiple/203000

3) Calculate the labeling ratio: moles of dye bound per mole of protein = A650 × dilution factor / (239000 × protein molar concentration)

Reactive dye stability and storage

Please note: the unopened powder can be stored for 6 months in the dark and dry at -20℃. The aqueous solution can not be stored when used. Any dissolved dye is best used immediately; anhydrous DMSO solution is stored at -20°C for up to 2 weeks.