- Description
- Additional Information
- Readable Documents
- Assay Principle
- Reviews
Key Benefits
- Cell permeable.
- No wash homogenous assay.
- Adaptable to High throughput assay platforms .
- Real time detection of NO activity.
- Applications – Flow Cytometry, Fluorescence Plate Reader, Fluorescence Microscopy
Additional information
| Kit Size | 0.125mg, 0.25mg |
|---|
Diaminofluorescein –2 Diacetate (DAF-2DA) is a non-fluorescent cell permeable reagent that can measure free Nitric Oxide (NO) and nitric oxide synthase (NOS) activity in living cells under physiological conditions. Once inside the cell the diacetate groups on the DFA-2DA reagent arehydrolyzed by cytosolic esterases thus releasing FAD-2 and sequestering the reagent inside the cell. Production of nitric oxide converts the non-fluorescent dye, DAF-2, to its fluorescent triaole derivative, DAF-2T.
DAF-2T can be observed by: excitation 488nm and measuring emission at 515nm.

Fig. 1. DAF-2DA, the non-fluorescent probe, enters the cells where cytosolic esterases hydrolysis the diacetate groups, releasing the non-fluorescent DAF-2 dye. DAF-2 then reacts with NO and O2 to produce DAF-2T, its triazole fluorescent derivative.

Fig. 2. Phase Contrast and fluorescent microscopic images of rat aorta-derived vascular smooth muscular cells loaded with DAF-2DA.
| Document Title |
| NOSProtocol |
| NOS Datasheet |
| msds.NOS |
| Title | File | Link | Author(s) | Journal | Year; Edition:Pages |
| Red wine consumption improves in vitro migration of endothelial progenitor cells in young, healthy individuals | Am J Clin Nutr 2010 92: 1 | July 2010; 161-169 |
| Reference |
| Direct evidence of NO production in rat hippocampus and cortex using a new fluorescent indicator: DAF-2 DA: H. Kojima, et al.; Neuroreport 9, 3345 (1998) Abstract |
| Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins: N. Nakatsubo, etal.; FEBS Lett. 427, 263 (1998) Abstract |
| Nitric Oxide Ameliorates Hydrophobic Bile Acid-induced Apoptosis in Isolated Rat Hepatocytes by Non-mitochondrial Pathways: E. Gumpricht, et al.; J. Biol.Chem. 277, 25823 (2002) Full Text; http://www.jbc.org/cgi/content/full/277/28/25823#SEC1 |
| Development of a fluorescent indicator for nitric oxide based on the fluoroscein chromophore: H. Kojima, et al.; Chem. Pharm. Bull. 46, 373 (1998)Abstract |
| Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins: H. Kojima, et al.; Anal. Chem. 70, 2446 (1998) Abstract |
| Part# | Reagent | Temperature |
| Part# 4006 | DAF-2DA, 1 Vial (NOS200-1) | 2-8C |
| Part# 4006 | DAF-2DA, 2 Vials (NOS200-2) | 2-8C |
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逆转录(reverse transcription)是以RNA为模板合成DNA的过程,即RNA指导下的DNA合成。此过程中,核酸合成与转录(DNA到RNA)过程与遗传信息的流动方向(RNA到DNA)相反,故称为逆转录。逆转录过程是RNA病毒的复制形式之一,需逆转录酶的催化。 逆转录过程的揭示是分子生物学研究中的重大发现,是对中心法则的重要修正和补充。人们通过体外模拟该过程,以样本中提取的mRNA为模板,在逆转录酶的作用下,合成出互补的cDNA,构建cDNA文库,并从中筛选特异的目的基因。该方法已成为基因工程技术中最常用的获得目的基因的策略之一。
1.模板提取(一般为RNA):Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定:分光光度计或NanoDrop
3.逆转录:逆转录试剂盒(或者一步法试剂盒),这一步可以用普通PCR做,也可以用水域做。
4.荧光定量PCR试剂:通常有用SYBR Green Mix做的,但是这里建议你用EvaGreen做,灵敏度和平行性都要好于SYBR Green,并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他:除了以上的那些还需要离心管、PCR管或板(Axygen反应比较好)、移液枪等,暂时就想到这么多。
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大家miRNA逆转录和qPCR的试剂盒用的是那个公司的啊,求推荐!

