| Monitoring the Targeting of Cathepsin D to the Lysosome by Metabolic Labeling and Pulse-chase Analysis
|
|
Author:
Date:
2017-11-05
[Abstract] Mannose 6-phosphate receptors function can be studied in living cells by investigating alterations in processing and secretion of their ligand Cathepsin D. The assay described here is well established in the literature and comprises the metabolic labeling of newly synthesized proteins with [35S] methionine-cysteine in HeLa cells to monitor Cathepsin D processing through secretory pathway and secretion using immunoprecipitation, SDS-PAGE and fluorography.
[摘要] 通过研究其配体组织蛋白酶D的加工和分泌的改变，可以在活细胞中研究甘露糖-6-磷酸受体功能。在此描述的测定在文献中已经很好地确定，并且包括新合成的蛋白质的代谢标记，其中[35 [S]甲硫氨酸半胱氨酸，以监测组织蛋白酶D处理，通过分泌途径和分泌使用免疫沉淀，SDS-PAGE和荧光。
【背景】组织蛋白酶d（CATD）是一种溶酶体天冬氨酸蛋白酶由甘露糖-6-磷酸受体（M6PRs）排序在哺乳动物细胞中该传输它从反面高尔基体网络到内涵体/溶酶体（戈什等人，2003 ）。将CatD合成为前体蛋白（〜52kDa），其在溶酶体中被切割以产生中间体（〜48kDa）或成熟的溶酶体形式（〜34kDa）。微量的前体蛋白质也从生物合成途径分泌（Benes et al。，2008）。 catD的丰度可以使用几种方法来确定，例如基于免疫荧光的染色（Poole等人，1972），蛋白质印迹，荧光活性测定（Bewley等人， （Hirst等人，2009; Kametaka等人，2007; Tavares等人，2011）或用脉冲追踪分析进行代谢标记（Hirst等人，2009; Kametaka等人， ...
|
|
|
| Preparation of Primary Neurons from Rat Median Preoptic Nucleus (MnPO)
|
|
Author:
Date:
2013-12-05
[Abstract] Studying cell physiology is really important to understand their function and to determine action mechanisms taking place in these cells. Using brain slices can be sometime difficult to directly access to cells like neurons. Even if it more steps are needed to get cultured cells, this type of preparation allow a better access to neurons and provide a good way to study their basal properties. Here we describe a protocol of a short term primary neurons culture from MnPO, allowing to kept neurons in good condition to perform electrophysiological recordings.
[摘要] 研究细胞生理学对于了解其功能和确定在这些细胞中发生的作用机制是非常重要的。 使用脑切片可能有时难以直接访问像神经元这样的细胞。 即使需要更多的步骤来培养细胞，这种类型的制剂可以更好地接近神经元，并提供研究其基础性质的好方法。 在这里，我们描述了一种来自MnPO的短期原代神经元培养的方案，允许神经元保持良好的状态进行电生理记录。
|
|
|
| Cell Isolation of Spleen Mononuclear Cells
|
|
Author:
Date:
2013-05-05
[Abstract] This method allows you to isolate different subclass mononuclear cells, like B-cells, T cells, CD4+ and CD8+ T, from mouse spleen. By conjugating cells with specific antibodies and subsequently magnetic beads isolation, using the technique from Miltenyi, this allows a high purity.
[摘要] 这种方法允许你从小鼠脾脏中分离不同的亚类单核细胞，如B细胞，T细胞，CD4 +和CD8 + T。 通过使用来自Miltenyi的技术使细胞与特异性抗体缀合并随后磁珠分离，这允许高纯度。
|
|
|