Cy5 SE

产品简介

菁染料是性能优良的荧光标记染料，摩尔吸光系数在荧光染料中是最高的，其琥珀酰亚胺酯是最常用的脂肪氨基标记试剂，广泛用于蛋白、抗体、核酸及其他生物分子的标记和检测。通过改变次甲基链的长度, 可改变其荧光发射波长，每增加一个双键，按照Huoffman规则正好红移约100nm。

菁染料Cy3和Cy5已成为基因芯片的首选荧光标记物;另外,Cy5, Cy5.5和Cy7的吸收在近红外区背景非常低，是荧光强度最高、最稳定的长波长染料。特别适合于活体小动物体内成像代替放射性元素。

产品参数

Ex(nm) 646    Em(nm) 664    Solvent: Water or DMSO

菁染料琥珀酰亚胺酯（CyDye NHS）的标记

菁染料和生物分子的比例F/P=4～12之间荧光强度最高，F/P值过高荧光探针会自我淬灭并影响生物分子的生物活性，标记生物分子最好是用单琥珀酰亚胺酯，但是用双修饰的CyDye NHS并没有发现交联。CyDye NHS标记抗体在pH (8.5～9.4)时10分钟F/P可达5～6，而在pH 7.0几乎不反应。我们用不同比例的Cy3标记anti-glutathione-S-transferase (GST)多克隆抗体发现用1:1, 5:1, 10:1 和20:1标记时得到的F/P值分别是0.28:1, 1.16:1, 2.3:1 和4.6:1。

操作步骤

水溶性Cy5 NHS标记(D-ser2)-leucineenkephalin 

1. Cy5 NHS 1.0 mg 溶解于400 μL DMSO后，加入到1mL 的玻璃瓶中盛有(D-ser2)-leucine-enkephalin acetate (YSGFLT, 0.75 mg) 的400 μL DMSO溶液。（Dye 和peptide 投料比是1:1）

2. 然后加入15 μL三乙胺，常温避光搅拌反应混合物过夜。

3. 用HPLC纯化蛋白，使用蛋白C18柱子(25 cm×10 mm)，每次上样注入2×400 μL，30分钟梯度洗脱从0.1% TFA水溶液到MeCN∶H2O（0.1% TFA）=70∶30，流速4mL /min。（对不同的蛋白选择不同的合适HPLC梯度流动相）

4. 收集适当的色带峰，标记多肽的保留时间比未标记的多肽长。

5. 产品冷冻干燥成粉末或在水溶液中，-20℃避光储存；必要时可用质谱表征。

6. CyDye标记的蛋白稳定性取决于蛋白本身。例如标记的IgG在4 ℃可避光保存2月；更长期的保存需加入等体积的甘油-20 ℃避光保存。

F/P计算

Cy5 在650 nm 的摩尔吸光系数为250000 M-1cm-1，所用蛋白在280 nm处的摩尔吸光系数为170000 M-1cm-1；Cy5在280nm处的吸收是650nm处的5%。按以下公式计算F/P值。

 [Cy5 dye] = (A650)/250000

 [peptide] =[A280-(0.05×A650)]/170 000

F/P final= [dye]/[peptide]= {0.68×A650}/ {A280- (0.05×A650)}

Product Introduction

Cyanine dye is a fluorescent labeling dye with excellent performance. Its molar absorption coefficient is the highest among fluorescent dyes. Its succinimidyl ester is the most commonly used aliphatic amino group labeling reagent and is widely used for labeling proteins, antibodies, nucleic acids and other biological molecules. And detection. By changing the length of the methine chain, the fluorescence emission wavelength can be changed. For each double bond added, the red shift is exactly 100 nm according to the Huoffman rule.

The cyanine dyes Cy3 and Cy5 have become the first choice fluorescent markers for gene chips; in addition, the absorption of Cy5, Cy5.5 and Cy7 is very low in the near-infrared region, and they are the long-wavelength dyes with the highest fluorescence intensity and the most stable. It is especially suitable for in vivo imaging of small living animals instead of radioactive elements.

Product parameter

Ex(nm) 646      Em(nm) 664     Solvent: Water or DMSO

Labeling of cyanine dye succinimide ester (CyDye NHS)

The ratio of cyanine dye and biomolecule is between F/P=4～12. The fluorescence intensity is the highest. If the F/P value is too high, the fluorescent probe will self-quench and affect the biological activity of biomolecules. It is best to use single amber to label biomolecules. Imide ester, but CyDye NHS modified with double did not find cross-linking. CyDye NHS-labeled antibody can reach 5-6 F/P in 10 minutes at pH (8.5-9.4), but hardly reacts at pH 7.0. We used different ratios of Cy3 to label anti-glutathione-S-transferase (GST) polyclonal antibodies and found that the F/P values ​​obtained when labeled with 1:1, 5:1, 10:1 and 20:1 were 0.28:1 , 1.16:1, 2.3:1 and 4.6:1.

Steps

Water-soluble Cy5 NHS labeling (D-ser2)-leucine enkephalin

1. After dissolving 1.0 mg of Cy5 NHS in 400 μL DMSO, add it to a 1 mL glass bottle containing 400 μL DMSO solution of (D-ser2)-leucine-enkephalin acetate (YSGFLT, 0.75 mg). (The feed ratio of Dye and peptide is 1:1)

2. Then add 15 μL of triethylamine, and stir the reaction mixture overnight at room temperature in the dark.

3. Purify the protein by HPLC, use a protein C18 column (25 cm×10 mm), inject 2×400 μL each time, 30 minutes gradient elution from 0.1% TFA aqueous solution to MeCN: H2O (0.1% TFA) = 70 ：30, flow rate 4mL/min. (Choose different suitable HPLC gradient mobile phases for different proteins)

4. Collect the appropriate color band peaks. The retention time of the labeled peptide is longer than that of the unlabeled peptide.

5. The product is freeze-dried into powder or stored in an aqueous solution at -20°C and protected from light; if necessary, it can be characterized by mass spectrometry.

6. The stability of CyDye-labeled protein depends on the protein itself. For example, labeled IgG can be stored in the dark at 4°C for 2 months; for longer-term storage, an equal volume of glycerol should be added to -20°C in dark storage.

F/P calculation

The molar absorption coefficient of Cy5 at 650 nm is 250 000 M-1cm-1, and the molar absorption coefficient of the protein used at 280 nm is 170 000 M-1cm-1; the absorption of Cy5 at 280 nm is 5% of that at 650 nm. Calculate the F/P value according to the following formula.

[Cy5 dye] = (A650)/250 000

[Peptide] =[A280-(0.05×A650)]/170 000

F/P final= [dye]/[peptide]= {0.68×A650}/ {A280- (0.05×A650)}