| Extraction and Measurement the Activities of Cytosolic Phosphoenolpyruvate Carboxykinase (PEPCK) and Plastidic NADP-dependent Malic Enzyme (ME) on Tomato (Solanum lycopersicum)
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Author:
Date:
2014-05-05
[Abstract] A recent study demonstrated that cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and NADP-malic enzyme (NADP-ME) have an important role in malate metabolism during fruit ripening (Osorio et al., 2013). PEPCK catalyze the ATP-dependent decarboxylation of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and NADP-ME, the reversible conversion of malate and pyruvate. Here, we present the detailed protocols to measure PEPCK activity in carboxylation direction by following oxidation of NADH and to measure NADP-ME activity based upon the reduction of NADP+.
[摘要] 最近的研究表明胞质磷酸烯醇丙酮酸羧激酶（PEPCK）和NADP-苹果酸酶（NADP-ME）在果实成熟期间在苹果酸代谢中具有重要作用（Osorio等人，2013）。 PEPCK催化草酰乙酸（OAA）向磷酸烯醇丙酮酸（PEP）和NADP-ME的ATP依赖性脱羧，苹果酸和丙酮酸的可逆转化。 在这里，我们提出详细的协议，以测量PEPCK活性的羧化方向通过以下氧化NADH和基于NADP + 的还原测量NADP-ME活性。
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| Immunoplaque Assay (Influenza Virus)
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Author:
Date:
2013-11-05
[Abstract] Despite developed long time ago, plaque assay is still the gold standard for viral titer quantification in modern virology. The standard crystal violet-based plaque assay relies on virus’ ability to induce cytopathic effect (CPE) which limits the assay to lytic viruses. Alternative viral quantification assays such as 50% tissue culture infectious assay (TCID50) and genetic material quantification by Q-PCR provide a different way of viral quantification with their own shortcoming. In here, we modified the fluorescent focus assay and developed an antibody-based immunoplaque assay which provides a reliable and reproducible viral quantification independent of CPE. Our assay not only allows accurate determination of viral titer, but also provides information on viral kinetics, ...
[摘要] 尽管发展很久以前，斑块测定仍然是现代病毒学病毒滴度量化的黄金标准。 标准的基于结晶紫的斑块测定依赖于病毒诱导细胞病变效应（CPE）的能力，其限制了对裂解病毒的测定。 替代性病毒定量测定如50％组织培养感染测定（TCID 50）和通过Q-PCR的遗传物质定量提供了具有其自身缺点的病毒定量的不同方式。 在这里，我们修改荧光焦点测定和开发基于抗体的免疫斑检测提供可靠和可重复的病毒定量独立于CPE。 我们的测定不仅允许病毒滴度的精确测定，而且提供关于病毒动力学，遗传稳定性和病毒种群的纯度的信息。
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