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NO合酶活性的测定
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| 本人要测单体化合物对NO合酶活性的影响，看了些相关研究，但还是不太明白。希望有做过的好心人给我指导下。南京建成有NO合酶试剂盒买，但好像是测其含量，并不是真正反映物质对酶活性的影响，不知道我的理解对不，是不是有其他的试剂盒可以测定，恳请各位大侠帮助。 |
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qingfengwu
金虫 (小有名气)
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- 性别: GG
- 专业: 神经生物学
Calbiochem的kit可以测。
★ ★ ★
小木虫(金币+0.5):给个红包，谢谢回帖交流
1949stone(金币+2,VIP+0): 11-30 16:15
小木虫(金币+0.5):给个红包，谢谢回帖交流
1949stone(金币+2,VIP+0): 11-30 16:15
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Nitric oxide synthase (NOS) has been found to play many diverse physiological roles ranging from neurotransmitter to vasodilator to cytotoxic agent. It has been implicated in many normal and pathophysiological processes. Three isoforms of NOS exist, though all three isoforms are present in many different tissues and cell types. The enzyme synthesizes nitric oxide (NO) during the conversion of L-arginine to L-citrulline in the presence of oxygen and the cofactors NADPH, FAD, FMN and BH4. Determining the activity of NOS and thus, the amount of NO produced in cells or tissues is rapidly becoming a commonly used technique in many areas of research. Several companies have developed colorometric and fluormetric NOS activity assays which measure the concentration of nitrate and nitrite. These assays are based on the fact that NO is converted to nitrate and nitrite; therefore, measuring the concentrations of these molecules is a measure of the total NO in the cell or tissue. These types of assays work well; however, they are an indirect measure of NOS activity and have detection limits of about 2.5 uM. NOS activity can be directly measured by quantifying the production of citrulline from arginine. The Calbiochem NOS Activity Assay Kit is based on the conversion of arginine to citrulline, using arginine labeled with 3H or 14C. Calbiochem claims the kit can detect down to the picomole level. The kit can be used for cell or tissue extracts and comes with enough spin columns for 50 reactions. The kit contains all reagents except for the radiolabeled arginine. The sample is mixed with equilibrated resin which binds to un-reacted arginine and is then applied to spin cups, allowing citrulline to elute through the column completely. The NOS activity is then determined by quantifying the radioactivity in the eluate with a liquid scintillation counter. The advantage of using this kit is that the spin cups replace column chromatography, which is traditionally used to separate arginine from citrulline. These columns are time consuming to prepare and use. Thus, a simple spin column to which the resin and sample is applied and centrifuged for 30 seconds is a major advantage. We were measuring NOS activity in fibroblasts and HUVEC (human umbilical vein endothelial cells). We purchased several of these kits and have processed over 200 samples, each consisting of one 10 cm plate of 100% confluent cells. The kit protocol says to use 100 ul equilibrated resin per sample. We found that this was not enough to bind all the unreacted arginine and started using 300 ul equilibrated resin per sample. Though the kit came with enough reagents for 50 reactions, using more resin per sample allowed us to process only 16 reactions per kit. As the kit is expensive to start with, this necessary modification made the kit too expensive to use. Rat cerebellum extract is provided in the kit as a positive control. No activity was detected in any of the rat cerebellum extract samples from any of the kits, even when thawed within 3 minutes of analysis. Purified NOS worked well as a positive control and verified the reaction was working. Overall, we were not able to detect NOS in our samples using this kit, even though Calbiochem claims it detects to the picomole level. This may be due to the fact that we have medium to low levels of NOS in our samples; however, we were surprised that even after combining six 10 cm plates of cells into one sample, we still did not observe activity. We were, however, able to detect NOS activity in our samples by fluorescence microscopy using DAF-2/DA (Calbiochem) in which the intensity of the fluorescence indicated a high level of activity, although it was not quantified using this method. We also measured expression of NOS in these cells using Applied Biosystems TaqMan® Gene Expression Assays specific for the three isoforms of NOS. We found NOS II was expressed at approximately 103 copies/ul and NOS III was expressed at approximately 105 copies/ul. Although expression cannot be directly related to activity, the positive results from these other experiments indicate possible problems with this kit. |
2楼2009-11-30 15:46:41
qingfengwu
金虫 (小有名气)
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国内的也有，你可以看看。一氧化氮合成酶检测试剂盒
★
1949stone(金币+1,VIP+0): 11-30 16:15
1949stone(金币+1,VIP+0): 11-30 16:15
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一氧化氮合成酶检测试剂盒(Nitric Oxide Synthase Assay Kit)可以检测活细胞内总的一氧化氮合成酶的活性。如果和特异性的一氧化氮合成酶抑制剂配合使用，也可以检测不同类型的一氧化氮合成酶的活性。 本试剂盒提供了一种在生理条件下通过荧光检测活细胞内一氧化氮合成酶活性的方法，不需要使用传统方法所需的放射性同位素。本试剂盒采用了可以穿透细胞膜的最新一代一氧化氮荧光检测探针DAF-FM DA(3-amino,4-aminomethyl-2',7'-difluorescein, diacetate)，在提供充足的底物的条件下检测细胞内的一氧化氮合成酶可以催化产生的一氧化氮量，从而检测出一氧化氮合成酶的活性。详细的检测原理参考图1。采用一些一氧化氮合成酶的抑制剂则可以测定出特定类型的一氧化氮合成酶的活性。例如，采用iNOS抑制剂，未加iNOS抑制剂测定出来的酶活力减去加了iNOS抑制剂测定出来的酶活力就是iNOS的酶活力。 本试剂盒是目前为止世界上最先进的一种一氧化氮合成酶检测试剂盒。和Calbiochem等多年前就开始提供的一氧化氮合成酶检测试剂盒相比无需使用同位素，和Sigma等最近推出的基于荧光的一氧化氮合成酶检测试剂盒相比采用了最新一代的荧光探针，灵敏度更高，特异性更好。本试剂盒的缺点是和其它荧光法一氧化氮合成酶检测试剂盒一样，仅能提供一氧化氮合成酶的相对活性。 本试剂盒和检测细胞内一氧化氮水平的不同之处在于提供了充足的底物L-Arginine和一些可以穿透细胞膜的反应辅助因子，确保一氧化氮合成酶的催化的一氧化氮合成不会受底物的量或辅助因子的量的限制，从而可以测定出细胞内一氧化氮合成酶活力。 本试剂盒最适合用于贴壁的细胞或组织的一氧化氮合成酶的检测，对悬浮细胞也可以进行检测。 本试剂盒可以测定100个样品。 http://www.beyotime.com/NO/s0025.htm |
3楼2009-11-30 15:48:33
4楼2009-12-01 10:07:42
