Aladdin Fluo™ 647 C₂-maleimide

Aladdin Fluo™ 是一种明亮的远红外荧光染料，其激发光谱非常适合于 594 nm 或 633 nm 激光谱线。Aladdin Fluo™ 647 染料用于成像和流式细胞分析中稳定信号的生成，具有水溶性和 pH 值不敏感性 (pH 4 - pH 10)。除反应性染料制剂外，我们还提供可与多种抗体、肽、蛋白、示踪剂和扩增底物偶联并且针对细胞标记和检测进行优化的 Aladdin Fluo™ 647 染料。 Aladdin Fluo™ 647 的马来酰亚胺衍生物是将该染料与蛋白、寡核苷酸硫代磷酸盐或低分子量配体上的硫醇基团偶联的较常用工具。所得 Aladdin Fluo™ 647 偶联物显示出比其他光谱相似荧光基团的偶联物更亮的荧光和更高的光稳定性。 关于该 Aladdin Fluo™ 马来酰亚胺的详细信息： 荧光基团标记：Aladdin Fluo™ 647 染料 反应性基团：马来酰亚胺 反应性：蛋白质和配基上的硫醇基团、寡核苷酸硫代磷酸盐 偶联物的 Ex/Em：651/671 nm 消光系数：265,000 cm-1M-1 光谱类似染料：Cy5 分子量：∼1250 典型偶联反应 在适当的缓冲液（10-100 mM 磷酸盐、Tris 或 HEPES）中，在 pH 值为 7.0-7.5 的环境下该蛋白的溶解浓度应为 50-100 µM。在此 pH 值范围内，蛋白硫醇基团亲核性强，在存在不计其数的蛋白胺（这些蛋白胺经过质子化，并且相对不具反应性）时，几乎仅与试剂反应。我们建议此时使用达到 DTT 或 TCEP 等还原剂摩尔量10倍的浓度还原所有二硫键。必须通过透析去除过量的 DTT，应在无氧条件下进行后续硫醇修饰，以防止二硫键再形成；在马来酰亚胺偶联之前使用 TCEP 时，无需采取这些预防措施。 Aladdin Fluo™ 马来酰亚胺通常在即将使用前溶于高质量无水二甲亚砜 (DMSO) 中，浓度为1-10 mM，储备液应尽可能避光储存。通常，将这种储备液逐滴加入蛋白溶液中，同时搅拌，产生大约10-20摩尔试剂/摩尔蛋白，反应在室温下进行2小时，或在 4°C 下过夜避光进行。通过添加过量的谷胱甘肽、巯基乙醇或其他可溶性低分子量硫醇，可以消耗任何未反应的硫醇反应性试剂。 偶联物纯化 通常使用凝胶过滤柱（如 Sephadex™ G-25、BioGel™ P-30 或等效物）将标记抗体与游离 Aladdin Fluo™ 染料分离。对于更大或更小的蛋白，选择具有适当分子量滤除点的凝胶过滤介质或通过透析纯化。我们提供了多种优化的纯化试剂盒，可用于不同量抗体偶联物： 0.5-1 mg 用抗体偶联物纯化试剂盒 20-50 µg 用抗体偶联物纯化试剂盒 50-100 µg 用抗体偶联物纯化试剂盒 Alexa Fluor™ 647 is a bright, far-red fluorescent dye with excitation ideally suited for the 594 nm or 633 nm laser lines. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor™ 647 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor™ 647 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more). The maleimide derivative of Alexa Fluor™ 647 is the most popular tool for conjugating the dye to a thiol group on a protein, oligonucleotide thiophosphate, or low molecular weight ligand. The resulting Alexa Fluor™ 647 conjugates exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores. Detailed information about this AlexaFluor™ maleimide: Fluorophore label: Alexa Fluor™ 647 dye Reactive group: maleimide Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates Ex/Em of the conjugate: 651/671 nm Extinction coefficient: 265,000 cm-1M-1 Spectrally similar dyes: Cy5 Molecular weight: ∼1250 Typical Conjugation Reaction The protein should be dissolved at a concentration of 50-100 µM in a suitable buffer (10-100 mM phosphate, Tris, or HEPES) at pH 7.0-7.5. In this pH range, the protein thiol groups are sufficiently nucleophilic that they react almost exclusively with the reagent in the presence of the more numerous protein amine groups, which are protonated and relatively unreactive. We recommend reducing any disulfide bonds at this point using a 10-fold molar excess of reducing agent such as DTT or TCEP. Excess DTT must be removed by dialysis and subsequent thiol-modification should be carried out under oxygen-free conditions to prevent reformation of the disulfide bonds; these precautions are not necessary when using TCEP prior to maleimide conjugation. The Alexa Fluor™ maleimide is typically dissolved in high-quality anhydrous dimethylsulfoxide (DMSO) at a concentration of 1-10 mM immediately prior to use, and stock solutions should be protected from light as much as possible. Generally, this stock solution is added to the protein solution dropwise while stirring to produce approximately 10-20 moles of reagent per mole of protein, and the reaction is allowed to proceed at room temperature for 2 hours or at 4°C overnight, protected from light. Any unreacted thiol-reactive reagent can be consumed by adding excess glutathione, mercaptoethanol, or other soluble low molecular weight thiol. Conjugate Purification Labeled antibodies are typically separated from free Alexa Fluor™ dye using a gel filtration column, such as Sephadex™ G-25, BioGel™ P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate: Antibody Conjugate Purification Kit for 0.5-1 mg () Antibody Conjugate Purification Kit for 20-50 µg () Antibody Conjugate Purification kit for 50-100 µg () Learn More About Protein and Antibody Labeling We offer a wide selection of Molecular Probes™ antibody and protein labeling kits to fit your starting material and your experimental setup. See ourAntibody Labeling kits or use ourKits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes™ Handbook. We’ll Make a Custom Conjugate for You If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Ourcustom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

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