
Product Name | SensoLyte ® 520 Neprilysin Activity Assay Kit *Fluorimetric* |
Size | 1 kit |
Catalog # | AS-72223 |
US$ | $526 |
Neprilysin (NEP) is a transmembrane metallopeptidase normally expressed by a variety of tissues. It is also known as neutral endopeptidase, and enkephalinase.NEP cleaves peptides at the N-terminal side of hydrophobic amino acid residues and is responsible for the degradation and inactivation of a variety of physiological substrates. NEP is a major extracellular amyloid beta-peptide degrading enzyme in the brain and targeting NEP is considered a potential therapeutic strategy for the prevention and treatment of Alzheimer"s disease. NEP has also been implicated in the pathogenesis of hypertension, diabetes, and cancer. The SensoLyte® 520 Neprilysin Assay Kit employs a novel internally quenched 5-FAM/QXL® FRET substrate for the detection of neprilysin activity.The enzyme cleaves the FRET substrate into two separate fragments resulting in the release of 5-FAM fluorescence, which can be monitored at excitation /emission= 490/520 nm. The long wavelength fluorescence of 5-FAM is less interfered by the autofluorescence of components in biological samples and test compounds. The assay can detect as low as 0.78 ng/mL of active Neprilysin. The Kit contains: 5-FAM/QXL® 520 Neprilysin substrate, Ex/Em=490/520 nm upon cleavage 5-FAM Fluorescence reference standard, Ex/Em=490/520 nm Recombinant human Neprilysin 2X Assay BufferInhibitorKit size: 100 assays RELATED PRODUCTS: SensoLyte® 520 β-Secretase Activity Assay *Fluorimetric*SensoLyte® 520 BACE2 Activity Assay *Fluorimetric*SensoLyte® 520 ADAM10 Activity Assay Kit *Fluorimetric*SensoLyte® 520 Neprilysin Activity Assay Kit *Fluorimetric*SensoLyte® Cathepsin Assays *Fluorimetric* SensoLyte® Anti-α-Synuclein (Human) ELISA Kit and α-Synuclein Recombinant Proteins SensoLyte® Anti-β-Amyloid (1-40) & (1-42) Mouse/ Rat ELISA Kits Recombinant human ß-Synucleinβ-Amyloid Peptides - Wide Selection Product Spotlight: Tau Products - Peptides and Recombinant Proteins Figure 1. Sensitivity of the assay has been tested using serial dilutions of recombinant human neprilysin. 5-FAM/QXL® 520 FRET substrate was incubated with the indicated amount of enzyme and fluorescence was measured after 60 min (FlexStation 384II, Molecular Devices). This assay can detect as low as 0.78 ng/mL of active neprilysin.Figure 2. Inhibition of neprilysin activity by Thiorphan as measured with SensoLyte® 520 Neprilysin Assay Kit. | |
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二、限制性内切酶消化样品DNA
三、凝胶电泳分离消化产物
四、如果靶序列>5kb,在0.25M的HCl中进行震荡脱嘌呤10min,ddH2O洗一次
五、用变性液震荡处理30min,ddH2O洗一次
六、中和液震荡处理30min
七、裁取合适大小的尼龙膜或硝酸纤维素膜进行转膜操作,可用真空转膜仪或者搭滤纸桥,需要20×SSC
八、制备探针,可用地高辛标记(以地高辛为例)
九、将膜放入杂交瓶,42°C预杂交30min
十、倒掉预杂交液,加入杂交液,适当的温度进行杂交4h至过夜
十一、洗膜,先用2×SSC+0.1%SDS,20-25°C洗2×5min,再用0.5×SSC+0.1%SDS洗2×15min。然后用washing buffer洗1-5min,接着用blocking solution洗30min后,用antibody solution洗30min,再用washing buffer洗2×15min,再用detection buffer洗2-5min后,取出膜,放于保鲜膜上,在结合有DNA的一面滴加CSPD ready-to-use后,立刻盖上保鲜膜,让CSPD ready-to-use均匀的布满膜表面,室温放置5min后,37°C温育10min以上
十二、放射自显影,可用成像系统信号累积模式显影或用X-ray显影

