描述：
DyLight™ 350 Fluorescent Properties: Excitation: 356nm, Emission: 423nm.
DyLight™350 Phalloidin允许研究人员通过将phalloidin结合F-肌动蛋白荧光标记固定细胞的细胞骨架。由于phalloidin的毒性，这个产品并不适用于活细胞。重构后的试剂提供了足够的材料在1:10稀释，100ul体系的情况下完成50个反应。DyLight™350荧光性质:激发光:356nm,发射光:423nm。存储:这种材料以冻干粉的形式可以在-20°C保存一年,干燥并避光。Phalloidin配制应该用纯甲醇制成储存液,详情请参考使用说明。一旦用纯甲醇，可在-20°C中保存一年,干燥并避光。溶于水之后稳定性降低，但与细胞孵育时必须溶于水。为了制成10倍的储存液, 在1.5毫升纯甲醇中重新冻干物质。用4%不含甲醇的甲醛固定细胞15分钟,然后用PBS冲洗三次，每次5分钟。一旦固定,请根据CST公司的指南进行细胞免疫荧光。一抗，二抗孵育过后,DyLight™650 Phalloidin可以用PBS1：10稀释(10ul每100µl测定体积)并添加到细胞中。让室温下孵育15分钟,然后用PBS冲洗一次。® Gold Antifade Reagent #9071封片并用合适的激发波长检查标本。< / p >Actin是一种广泛存在的真核蛋白，也是细胞骨架的重要组成部分。在哺乳动物中至少六个亚型。Nonmuscleβγ-actin,也称为胞质肌动蛋白,主要在非肌肉细胞中表达，控制细胞结构和动力(1)。Actin主要以纤维聚合物也叫F-actin的形式存在。它主要响应细胞骨架重组信号在胞质分裂等过程,内吞作用,或应激反应,cofilin促进分裂和f -肌动蛋白解聚作用导致增加单体的球状G-actin形式(2)。Phalloidin是存在于羊肚菌中的天然有毒二环肽, Amanita phalloides能迅速与f -肌动蛋白结合，亲和力强(3)。
原厂资料：
Description
DyLight™ 350 Phalloidin allows researchers to fluorescently label the cytoskeleton through the binding of phalloidin to F-actin. This product is not intended for use on live cells due to the toxicity associated with phalloidin. After reconstitution the stock solution provides enough material to perform 50 assays based on a 1:10 dilution and a 100 μl assay volume.
DyLight™ 350 Fluorescent Properties: Excitation: 356nm, Emission: 423nm.
Bioactivity
Storage: This material is provided as lyophilized solid that is stable for 1 year at -20°C, desiccated and protected from light.
Phalloidin conjugates should be reconstituted in pure methanol to make stock solutions, please refer to the directions for use for details. Once reconstituted in pure methanol, stock solutions are stable for 1 year at -20°C, desiccated and protected from light. Stability in aqueous solutions is low and the conjugate should only be in the presence of an aqueous solution during incubation with cells.
Directions for Use
To make a 10X stock, reconstitute the lyophilized material in 0.5 ml pure methanol.
Fix cells for 15 minutes using fresh, methanol-free 4% formaldehyde, then rinse three times in PBS for 5 minutes each. Once fixed, please follow Cell Signaling Technology protocol for immunostaining. Following incubation of the primary and secondary antibodies, DyLight™ 350 Phalloidin can be diluted 1:10 (10 µl per 100 µl assay volume) in PBS and added to the cells. Allow to incubate for 15 minutes at room temperature, then rinse once with PBS. Coverslip slides with ProLong® Gold Antifade Reagent #9071 and examine specimen using appropriate excitation wavelength.
Background
Actin, a ubiquitous eukaryotic protein, is the major component of the cytoskeleton. At least six isoforms are known in mammals. Nonmuscle β- and γ-actin, also known as cytoplasmic actin, are predominantly expressed in nonmuscle cells, controlling cell structure and motility (1). Actin exists mainly as a fibrous polymer, F-actin. In response to cytoskeletal reorganizing signals during processes such as cytokinesis, endocytosis, or stress, cofilin promotes fragmentation and depolymerization of F-actin resulting in an increase in the monomeric globular form, G-actin (2). Phalloidin is a natually occuring toxic bicyclic peptide found in the deathcap toadstool, Amanita phalloides, that rapidly binds to F-actin with strong affinity (3).
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