| Quantification of Protein Kinase A (PKA) Activity by An in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme
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Author:
Date:
2020-06-20
[Abstract] In order to acquire fertilizing potential, mammalian sperm must undergo a process known as capacitation, which relies on the early activation of Protein Kinase A (PKA). Frequently, PKA activity is assessed in whole-cell experiments by analyzing the phosphorylation status of its substrates in a western-blot. This technique faces two main disadvantages: it is not a direct measure of the kinase activity and it is a time-consuming approach. However, since PKA can be readily obtained from sperm extracts, in vitro assays such as the “radioactive assay” can be performed using the native enzyme. Unlike western-blot, the radioactive assay is a straightforward technique to evaluate PKA activity by quantification of incorporated 32P into a peptidic ...
[摘要] [Abstract]为了获得受精潜能，哺乳动物的精子必须经历一个被称为电容的过程，这个过程依赖于蛋白激酶A（PKA）的早期激活。通常，在全细胞实验中，通过分析其底物在western-blot中的磷酸化状态来评估PKA的活性。这种技术面临着两个主要的缺点：它不是对激酶活性的直接测量，而且是一种耗时的方法。然而，由于PKA可以很容易地从精子提取物中获得，体外检测如"放射性检测"可以使用原生酶进行。与western-blot不同的是，放射性测定法是一种直接的技术，通过将整合的32P定量到肽类底物中来评价PKA的活性。这种方法很容易允许分析PKA的不同激动剂或拮抗剂。由于小鼠精子是可溶性PKA的丰富来源，这种测定法可以进行简单的分馏，使PKA既可用于体外测试药物对PKA活性的影响，也可用于跟踪PKA活性在增容开始时的变化。
[Background] ...
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| Isolation and Culture of Human Adipose-derived Stem Cells from Subcutaneous and Visceral White Adipose Tissue Compartments
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Author:
Date:
2016-11-20
[Abstract] Human Adipose-derived Stem/Stromal Cells (ASCs) have been widely used in stem cell and obesity research, as well as clinical applications including cell-based therapies, tissue engineering and reconstruction. Compared with mesenchymal stem cells (MSCs) derived from other tissues such as umbilical cord and bone marrow, isolation of ASCs from human white adipose tissue (WAT) has great advantages due to its rich tissue source and simple surgical procedure. In this detailed protocol we describe a protocol to isolate and characterize ASCs from human WAT. Molecular characterization of isolated ASCs was performed through surface marker expression profiling using flow cytometry. Adipogenic capacity of the isolated ASCs was confirmed through inducing adipogenic differentiation and Oil Red O ...
[摘要] Human Adipose-derived Stem/Stromal Cells (ASCs) have been widely used in stem cell and obesity research, as well as clinical applications including cell-based therapies, tissue engineering and reconstruction. Compared with mesenchymal stem cells (MSCs) derived from other tissues such as umbilical cord and bone marrow, isolation of ASCs from human white adipose tissue (WAT) has great advantages due to its rich tissue source and simple surgical procedure. In this detailed protocol we describe a protocol to isolate and characterize ASCs from human WAT. Molecular characterization of isolated ASCs ...
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| cAMP Accumulation Assays Using the AlphaScreen® Kit (PerkinElmer)
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Author:
Date:
2013-07-05
[Abstract] Cyclic adenosine monophosphate (cAMP) is an intracellular signaling messenger derived from the catalytic conversion of ATP, and is a major product of activated Gs protein-coupled receptors. Conversely, formation of cAMP is inhibited by Gi protein-coupled receptors. This protocol has been optimized for the detection of ligand-mediated cAMP accumulation in adherent immortal cell lines expressing Gs-coupled receptors.
[摘要] 环腺苷一磷酸（cAMP）是来源于ATP的催化转化的细胞内信号传导信号，并且是活化的G蛋白偶联受体的主要产物。 相反，cAMP的形成受G 1 i蛋白偶联受体抑制。 该方案已经被优化用于检测表达G s - 偶联受体的贴壁无限增殖细胞系中配体介导的cAMP积累。
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