CY5
(Synonyms: 2-[5-[1-(5-羧基戊基)-1,3-二氢-3,3-二甲基-5-磺基-2H-吲哚-2-亚基]-1,3-戊二烯基]-1-乙基-3,3-二甲基-5-磺基-3H-吲哚内盐,Sulfo-Cyanine5) 目录号 : GC35769
CY5是一种含花青（Cyanine）的荧光染料，常用于蛋白、抗体以及小分子化合物的标记，最大激发/发射波长分别为650/670nm。
Cas No.:146368-11-8
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
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- Purity: >97.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
本方案仅提供一个指导，请根据您的具体需要进行修改。
1. 溶液配制
（1）工作液：用DMSO溶解CY5固体，终浓度为10mM，过滤除菌。
注意：工作液必须现配现用。使用前需先使用500μg/mL缩合液(货号：GA11126) 活化后方可进行后续标记实验。
2.使用CY5标记蛋白质的实验步骤
（1）蛋白准备：将蛋白溶解在不含伯胺（如Tris或甘氨酸）和铵离子的pH 8.5缓冲液中，浓度配制为2-10 mg/mL，以获得最佳标记效率。若pH低于8.0，用1 M碳酸氢钠进行调整。
（2）CY5工作液用量计算：标记反应所需的CY5(MW=656.81)用量取决于要标记蛋白的用量，CY染料与蛋白的最佳摩尔比为10:1左右。
（3）标记反应：取算好体积的CY5工作液缓慢加入到蛋白样品溶液中，轻轻摇匀混合，然后短暂离心将样品收集在反应管底部。切忌剧烈混匀，以防蛋白样品变性失活。
（4）孵育反应：将反应小管置于避光处，在室温条件下轻轻摇晃孵育60min。每隔10-15min，将反应小管轻轻颠倒几次，以充分混合两种反应物，提高标记效率。
注意：
（1）本品为未活化的荧光染料，使用本品标记多肽和蛋白等生物分子需先进行羧酸活化，如需活化形式推荐sulfo-Cyanine5 NHS Ester(货号:GC59174)。
（2）该方案为使用CY5用于蛋白质标记实验提供指导。可以根据其他文献和具体实验要求进行调整。操作应在无菌环境中进行，防止污染物干扰反应。避免直接接触反应试剂。
CY5 is a cyanine-containing fluorescent dye that is commonly used to label proteins, antibodies, and small molecule compounds. Its maximum excitation/emission wavelengths are 650/670nm, respectively[1]. The basic structure of CY5 is a cyclic compound with a conjugated double bond system. It has excellent fluorescence properties in the far-red region and good photostability[2]. CY5 usually exists in the form of its active ester or azide, which enables it to be labeled with biological molecules such as proteins and nucleic acids through covalent bonds[3]. The optimal molar ratio of CY5 to protein is about 10:1[4]. CY5 has a wide range of applications. It is not only suitable for biomarkers and cell imaging, but also for nucleic acid detection, protein analysis, and drug screening[5]. The molecular weight of this product is 656.81. It is for research purposes only and is not suitable for food, drugs, medical devices, or cosmetics.
References:
[1] Ferri G L, Isola J, Berger P, et al. Direct eye visualization of Cy5 fluorescence for immunocytochemistry and in situ hybridization[J]. Journal of Histochemistry & Cytochemistry, 2000, 48(3): 437-444.
[2] Yuan L, Lin W, Zheng K, et al. Far-red to near infrared analyte-responsive fluorescent probes based on organic fluorophore platforms for fluorescence imaging[J]. Chemical Society Reviews, 2013, 42(2): 622-661.
[3] Gerowska M, Hall L, Richardson J, et al. Efficient reverse click labeling of azide oligonucleotides with multiple alkynyl Cy-Dyes applied to the synthesis of HyBeacon probes for genetic analysis[J]. Tetrahedron, 2012, 68(3): 857-864.
[4] Yu H, Chao J, Patek D, et al. Cyanine dye dUTP analogs for enzymatic labeling of DNA probes[J]. Nucleic Acids Research, 1994, 22(15): 3226-3232.
[5] Ntziachristos V, Schellenberger E A, Ripoll J, et al. Visualization of antitumor treatment by means of fluorescence molecular tomography with an annexin V–Cy5. 5 conjugate[J]. Proceedings of the National Academy of Sciences, 2004, 101(33): 12294-12299.
CY5是一种含花青（Cyanine）的荧光染料，常用于蛋白、抗体以及小分子化合物的标记，最大激发/发射波长分别为650/670nm[1]。CY5的基本结构是一个带有共轭双键体系的环状化合物，在远红区具有优异的荧光特性，具有较好的光稳定性[2]。CY5通常以其活性酯或叠氮化物的形式存在，这使得它能够通过共价键与蛋白质、核酸等生物分子进行标记[3]。CY5与蛋白的最佳摩尔比为10:1左右[4]。CY5的应用范围非常广泛，不仅适用于生物标记和细胞成像，还可用于核酸检测、蛋白质分析以及药物筛选等领域[5]。本产品分子量为656.81，仅供研究用途，不适用于食品、药品、医疗器械或化妆品。
| Cas No. | 146368-11-8 | SDF | |
| 别名 | 2-[5-[1-(5-羧基戊基)-1,3-二氢-3,3-二甲基-5-磺基-2H-吲哚-2-亚基]-1,3-戊二烯基]-1-乙基-3,3-二甲基-5-磺基-3H-吲哚内盐,Sulfo-Cyanine5 | ||
| Canonical SMILES | CC(/C(N1CCCCCC(O)=O)=C\C=C\C=C\C2=[N+](CC)C(C=CC(S(=O)([O-])=O)=C3)=C3C2(C)C)(C)C4=C1C=CC(S(=O)(O)=O)=C4 | ||
| 分子式 | C33H40N2O8S2 | 分子量 | 656.81 |
| 溶解度 | Water: 25 mg/mL (38.06 mM) | 储存条件 | Store at -20°C,protect from light |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 1.5225 mL | 7.6126 mL | 15.2251 mL |
| 5 mM | 0.3045 mL | 1.5225 mL | 3.045 mL |
| 10 mM | 0.1523 mL | 0.7613 mL | 1.5225 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Labeling Antibodies with Cy5-Phycoerythrin
Cold Spring Harb Protoc 2019 Sep 3;2019(9).PMID:31481492DOI:10.1101/pdb.prot099317.
Conjugates of the FRET dye Cy5-phycoerythrin (Cy5PE) with antibodies are relatively straightforward to make. The protocol does require synthesis of the Cy5PE tandem dye. Phycoerythrin (PE) can be purchased from multiple vendors. This type of conjugate is useful for immunofluorescence studies involving protein targets with low expression levels. Although the entire conjugation can be performed in a single day, there is an overnight stopping point. When initially making Cy5PE derivatives, several different conjugates with varying ratios of CY5 to PE should be made. These should be tested by conjugating to a well-characterized antibody. Absorbance spectra readings are a very worthwhile step to determine the quality of the Cy5PE label.
Sequence-dependence of Cy3 and CY5 dyes in 3' terminally-labeled single-stranded DNA
Sci Rep 2022 Aug 31;12(1):14803.PMID:36045146DOI:10.1038/s41598-022-19069-9.
Fluorescence is an ideal tool to see and manipulate nucleic acids, and engage in their rich and complex biophysical properties. Labeling is the preferred approach to track and quantify fluorescence with nucleic acids and cyanine dyes are emblematic in this context. The fluorescent properties of cyanine dyes are known to be sequence-dependent, with purines in the immediate vicinity increasing the fluorescence intensity of Cy3 and CY5 dyes, and the ability of nucleobases to modulate the photophysical properties of common fluorophores may influence fluorescence measurements in critical assays such as FISH, qPCR or high-throughput sequencing. In this paper, we comprehensively map the sequence-dependence of Cy3 and CY5 dyes in 3'-fluorescently labeled single-stranded DNA by preparing the complete permutation library of the 5 consecutive nucleotides immediately adjacent to the dye, or 1024 sequences. G-rich motifs dominate the high fluorescence range, while C-rich motifs lead to significant quenching, an observation consistent with 5'-labeled systems. We also uncover GCGC patterns in the extreme top range of fluorescence, a feature specific to 3'-Cy3 and CY5 oligonucleotides. This study represents the final piece in linking nucleotide identity to fluorescence changes for Cy3, CY5 and fluorescein in all 3', 5', single-stranded and double-stranded DNA formats.
Development of Lipidoid Nanoparticles for siRNA Delivery to Neural Cells
AAPS J 2021 Dec 6;24(1):8.PMID:34873640DOI:10.1208/s12248-021-00653-2.
Lipidoid nanoparticles (LNPs) are the delivery platform in Onpattro, the first FDA-approved siRNA drug. LNPs are also the carriers in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While these applications have demonstrated that LNPs effectively deliver nucleic acids to hepatic and muscle cells, it is unclear if LNPs could be used for delivery of siRNA to neural cells, which are notoriously challenging delivery targets. Therefore, the purpose of this study was to determine if LNPs could efficiently deliver siRNA to neurons. Because of their potential delivery utility in either applications for the central nervous system and the peripheral nervous system, we used both cortical neurons and sensory neurons. We prepared siRNA-LNPs using C12-200, a benchmark ionizable cationic lipidoid along with helper lipids. We demonstrated using dynamic light scattering that the inclusion of both siRNA and PEG-lipid provided a stabilizing effect to the LNP particle diameters and polydispersity indices by minimizing aggregation. We found that siRNA-LNPs were safely tolerated by primary dorsal root ganglion neurons. Flow cytometry analysis revealed that CY5 siRNA delivered via LNPs into rat primary cortical neurons showed uptake levels similar to Lipofectamine RNAiMAX-the gold standard commercial transfection agent. However, LNPs demonstrated a superior safety profile, whereas the Lipofectamine-mediated uptake was concomitant with significant toxicity. Fluorescence microscopy demonstrated a time-dependent increase in the uptake of LNP-delivered CY5 siRNA in a human cortical neuron cell line. Overall, our results suggest that LNPs are a viable platform that can be optimized for delivery of therapeutic siRNAs to neural cells.
CY5 total protein normalization in Western blot analysis
Anal Biochem 2015 Oct 1;486:54-61.PMID:26095394DOI:10.1016/j.ab.2015.06.017.
Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on CY5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to CY5 total protein control signals over the whole loading range from 2.5 to 20.0μg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that CY5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence.
Cy3-Cy5 covalent heterodimers for single-molecule photoswitching
J Phys Chem B 2008 Sep 25;112(38):11878-80.PMID:18754575DOI:10.1021/jp806698p.
Covalent heterodimers of the Cy3 and CY5 fluorophores have been prepared from commercially available starting materials and characterized at the single-molecule level. This system behaves as a discrete molecular photoswitch, in which photoexcitation of the CY5 results in fluorescence emission or, with a much lower probability, causes the CY5 to enter into a long-lived, but metastable, dark state. Photoinduced recovery of the emissive CY5 is achieved by very low intensity excitation (5 W cm(-2)) of the Cy3 fluorophore at a shorter wavelength. A similar system consisting of proximal, but not covalently linked, Cy3 and CY5 has found application in stochastic optical reconstruction microscopy (STORM), a single-molecule localization-based technique for super-resolution imaging that requires photoswitching. The covalent Cy3-Cy5 heterodimers described herein eliminate the need for probabilistic methods of situating the Cy3 and CY5 in close proximity to enable photoswitching. As proof of principle, these heterodimers have been applied to super-resolution imaging of the tubular stalk structures of live Caulobacter crescentus bacterial cells.