在大腸桿菌與酵母菌蛋白質體晶片中量化其蛋白質的濃度;Quantification of protein concentration in E.coli and yeast proteome microarrays

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Title:	在大腸桿菌與酵母菌蛋白質體晶片中量化其蛋白質的濃度;Quantification of protein concentration in E.coli and yeast proteome microarrays
Authors:	李毓慈;Li, YU-TZU
Contributors:	生醫科學與工程學系
Keywords:	蛋白質體晶片;proteome microarray
Date:	2019-07-25
Issue Date:	2019-09-03 14:36:52 (UTC+8)
Publisher:	國立中央大學
Abstract:	蛋白質微陣列是一個高通量的生物感測器，他能夠提供大數據的蛋白質分析，也是探尋蛋白質相互作用的有力工具之一。在我們實驗室中，蛋白質晶片上點滿了酵母菌或大腸桿菌的蛋白質體。而這些基因庫蛋白質皆有穀胱甘肽S-轉移酶多組氨酸或組氨酸標記物，他們可與含有螢光的特定抗體辨識結合。我們可藉由電腦分析得到螢光訊號的強弱而分析抗體鍵結的相對數量。由於實驗是無法肉眼觀測，故晶片上有可能具有非特定鍵結的蛋白質影響實驗的準確性。因此我們需要從中做出可對比的劑量反應曲線以作為往後實驗的對照。我們從酵母菌基因庫中選出CRABP 1為特定對照蛋白；從大腸桿菌基因庫中選出nagA為特定對照蛋白。首先大量純化以得到高濃度的蛋白質，在稀釋成不同濃度並點在醛基晶片上測試，最終將特定對照蛋白與蛋白質體共同點在晶片上做出劑量反應曲線。而大量蛋白質點在一個小晶片上是非常難以控制的，在環境以及蛋白質活性的種種條件下，我們最終特定對照蛋白質濃度只能夠涵蓋基因庫約90%的蛋白質。其他過高濃度則無法判定。但此實驗對於未來的研究也提供一個對照。;Protein microarrays are high-throughput biosensors that provide protein analysis of big data and are one of the powerful tools for exploring protein interactions. In our laboratory, protein chip were printed with yeast or E.coli proteome. These gene library proteins have glutathione S-transferase polyhistidine (GST-His6)-tagged or His-tagged that bind to fluorescently recognized antibodies. We can analyze the intensity of the fluorescent signal by computer and calculate the relative amount of protein-antibody binding. Since the experiment is visually unobservable, proteins on the chip with non-specific binding may affect the accuracy of the experiment. Therefore, we need to make a comparable dose response curve as a control for subsequent experiments. We chose CRABP 1 as a specific control protein in the yeast gene library; and we selected nagA as a specific control protein from the E. coli gene library. First, we purified in large quantities to obtain high concentration of protein, then diluted to different concentrations and printed on the aldehyde chip, and finally a specific control protein is printed with the proteome to make a dose response curve on the chip. Although it is difficult to control large numbers of protein spots on small chip. Under the environment conditions and protein activity, we can only cover about 90% of the proteins, and other concentrations are too high to be determined. But this experiment also provides a comparison for future research.
Appears in Collections:	[Institute of Biomedical Engineering] Electronic Thesis & Dissertation

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