Caspase-3 is an active cell-death protease involved in the execution phase of apoptosis, during which cells undergo morphological changes such as DNA fragmentation, chromatin condensation and apoptotic body formation. Caspase-3 DEVD-R110 Fluorometric and Colorimetric Assay Kit provides a simple assay system for fast and highly sensitive detection of caspase-3 activity either by fluorescence or absorbance in mammalian cells. The fluorogenic and chromogenic substrate (Ac-DEVD)2-R110 contains two DEVD tetrapeptides and is completely hydrolyzed by the enzyme in two successive steps. Cleavage of the first DEVD peptide results in the monopeptide Ac-DEVD-R110 intermediate, which has absorption and emission wavelengths similar to those of R110 (λ_Ex/λ_Em = 496/520 nm) but has only about 10%of the fluorescence of the latter. Hydrolysis of the second DEVD peptide releases the dye R110, leading to a substantial fluorescence increase.

Although fluorometric detection of the end products is preferred because of the superior sensitivity, detection by absorbance is also possible. In fact, the extinction coefficient ofR110 is 10 times higher than that of p-nitroaniline (pNA), a dye commonly used in chromogenic substrates, making R110-based substrates significantly more sensitive than pNA-based substrates, even by colorimetric detection.

The assay kit includes Ac-DEVD-CHO, which is a caspase-3 inhibitor and can be used as a negative control. Also, R110 is provided in the kit for generating a standard curve, which can be used for quantifying caspase-3 activity.

Note: While caspase-3 preferentially cleaves the consensus sequence DEVD compared to other substrate sequences, other caspases also can cleave DEVD efficiently. Overlapping caspase substrate recognition limits the usefulness of caspase substrate peptides for distinguishing between different caspase activities in cell lysates.

Kit Components:

• Cell lysis buffer
• Assay buffer
• Enzyme substrate (Ac-DEVD)2-R110
• Enzyme inhibitor Ac-DEVD-CHO
• R110

Features:

• Fast: Fast enzyme kinetics.
• Sensitive: The enzymatic reaction forms intensely yellow colored and highly green fluorescent rhodamine 110 (R110) product.
• Versatile: Compatible with both fluorometric and colorimetric detection systems.

Biotium also offers the Caspase-3 DEVD-R110 Fluorometric HTS Assay Kit (catalog # 30009), a one step homogenous assay for high throughput screening (HTS) using fluorescent-based measurement. For real-time detection of caspase-3 activity in intact cells, see our novel NucView™488 Caspase-3 Substrate.

Biotium公司是一家在荧光技术领域独树一帜的生物技术公司，公司位于美国加利福尼亚的BAYAREA------世界上首要的生物科技中心。该公司一直致力于开发和生产用于生物医学研究的荧光指示剂及其他特殊生化产品。其产品被广泛应用于细胞生物学、蛋白质组学、分子生物学、免疫学、微生物学、诊断学、生物化学、神经科学、血液流动检测及大规模药物筛选等众多领域。Biotium公司专利产品众多，包括已经被广泛使用的实时PCR定量荧光染料EvaGreen、具有划时代意义的凝胶核酸染料GelRed和GelGreen，以及最新推出的CheetahTaq热启动酶个CF系列蛋白质标记染料等等。

GelReD®是一种超灵敏、极稳定和环境安全的荧光核酸染料，用于取代高毒性溴化乙锭（EB），用于在琼脂糖凝胶或聚丙烯酰胺凝胶中染色dsDNA、SSDN或RNA。GelRed®比EB敏感得多，无需拆解步骤。
GelReD®和EB具有几乎相同的光谱，因此您可以直接更换EB与GeldRead®，而不改变您现有的成像系统。
凝胶红可用于琼脂糖凝胶中dsDNA、ssDNA或RNA的预染色或后染色。凝胶红还可用于聚丙烯酰胺凝胶中dsDNA、ssDNA或RNA的后凝胶染色。因此，凝胶化与下游DNA操作（如限制性消化、测序和克隆）兼容。
优势

1.比EB更安全：Ames试验和其他试验显示无致突变和非细胞毒性

2.易处理：通过直接排放到下水道或常规垃圾中的环境安全试验

3.超灵敏：比EtBr和SYBR®安全型更灵敏

4.非常稳定：可在水中使用，在室温下长期储存稳定，可微波

5.使用简单：非常简单的预处理或电泳后凝胶染色程序

6.与标准紫外线透照仪兼容：无需改变光学设置即可替代EtBr

7.与下游应用兼容：凝胶纯化、限制性消化、测序和克隆

GelRed和GelGreen是设计以取代剧毒的代替高毒性的溴化乙锭（EtBr）的新一代核酸凝胶荧光染料。Biotium科学家研发的GelRed和GelGreen集低毒性、高灵敏性和出色的稳定性等优点于一体，效果由于EtBr及其他EtBr替代品。

几十年来，EtBr因极其低廉的价格，一般的灵敏度，一直被用作核酸凝胶染色的主要染料。然而EtBr是一个高度诱变的物质。该染料的使用安全隐患，净化及废物处置的相关费用等，终导致其代价高昂。正因如此，近年来市场上出现了一些替代品，如SYBR系列染料等。尽管这些替代染料减少了致突变性，但却丧失了染料其他方面的功能。例如，SYBRSafe的灵敏度很低，SYBRGreen及SYBRGold的稳定性远远低于EtBr。SYBR染料能快速的进入细胞，结合线粒体及细胞核DNA，是的染料在一定浓度是有毒的。事实上，在紫外线或其他诱变剂诱导下，SYBRGreenI强烈的突变性已经被广泛认知。（Ohta，etal.MutRes492,91(2001)）

为了保证GelRed和GelGreen的安全性，Biotium的科学家采用一种新型非常简单的概念：阻止染料进入活细胞减少遗传毒性。我们相信，不给DNA结合染料渗透活细胞中结合基因组DNA的机会，便可以保证染料没有或大幅度减少其诱变性作用。因此，我们设计的GelRed和GelGreen染料拥有独特化学结构保证其无法渗透细胞膜。Ames试验验证即使浓度远远高于实验者操作凝胶染色的工作浓度，GelRed和GelGreen也无诱变性。此外，环境的安全性试验表示，GelRed和GelGreen是完全无害的，对水生生物无毒。因此，废弃的GelRed和GelGreen可以直接倒入下水道，或按照普通垃圾处理。