| Analysis of Starch Synthase Activities in Wheat Grains using Native-PAGE
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Author:
Date:
2016-01-20
[Abstract] Starch synthases are one class of key enzymes involving in the synthesis of cereal starch, which transfer glucose from ADP-glucose to the non-reducing end of pre-existing α-(1-4)-liked glucosyl chains of amylopectin. This protocol is highly reproducible for assaying activities for starch synthase I and IIIa in wheat and barley endosperm at qualitative level and quantitative level. The protocol includes separating proteins isolated from developing endosperm with native-PAGE containing glycogen from oyster, incubating protein gels with ADP-glucose solution, and staining gels with iodine solution. The method allows researchers to compare the levels or changes of starch synthase activities.
[摘要] 淀粉合酶是涉及谷物淀粉合成的一类关键酶，其将葡萄糖从ADP-葡萄糖转移到预先存在的支链淀粉的α-（1-4） - 葡萄糖基链的非还原端。 这个协议是高度可重复的测定淀粉合成酶I和IIIa在小麦和大麦胚乳的定性水平和定量水平的活动。 该方案包括从发育的胚乳分离的蛋白质与来自牡蛎的含有糖原的天然PAGE，用ADP-葡萄糖溶液孵育蛋白质凝胶，并用碘溶液染色凝胶。 该方法允许研究人员比较淀粉合酶活性的水平或变化。
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| Immunoprecipitation of Proteins in Caenorhabditis elegans
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Author:
Date:
2015-04-05
[Abstract] Immunoprecipitation (IP) is a biochemical technique to precipitate a protein out of solution using an antigen that can specifically bind to that protein. IP can be performed to isolate and concentrate one particular protein from a sample of thousands of different proteins. IP is also readily performed to pull down interacting proteins of complexes out of solution. This protocol outlines the methods used to IP proteins in whole worm lysates and their preparation for detection on Western blots using denaturing conditions.
[摘要] 免疫沉淀（IP）是使用可以特异性结合该蛋白质的抗原将蛋白质从溶液中沉淀出来的生物化学技术。 IP可以从数千种不同蛋白质的样品中分离和浓缩一种特定蛋白质。 IP也容易进行以从溶液中取出复合物的相互作用蛋白。 该协议概述了用于IP蛋白在整个蠕虫裂解物中的方法及其用于使用变性条件在蛋白质印迹上检测的方法。
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| An Improved Method for PAGE-based Detection of Phosphorylated Protein in Yeast
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Author:
Date:
2012-06-20
[Abstract] One dimensional polyacrylamide gel electrophoresis has been successfully used to detect protein phosphorylation. This method is very simple and highly reproducible. Hyperhosphorylated proteins usually migrate slowlier than dephosphorylated proteins. However, not all phosphorylated proteins can be readily detected, due to sub-optimal sample preparation and electrophoresis conditions. Here, an improved method is described that can detect phosphorylation of yeast proteins ranging from 15 kD to 200 kD. The improvement in gel electrophoresis should also be applicable to mammalian culture cells.
[摘要] 已经成功地使用一维聚丙烯酰胺凝胶电泳来检测蛋白质磷酸化。 这种方法非常简单和高度可重现。 高磷酸化蛋白通常比去磷酸化蛋白更慢地迁移。 然而，由于次优的样品制备和电泳条件，不是所有的磷酸化蛋白都可以容易地检测。 这里，描述了一种改进的方法，其可以检测范围从15kD至200kD的酵母蛋白质的磷酸化。 凝胶电泳的改进也应适用于哺乳动物培养细胞。
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