Native antigen fractionation protein microarrays for biomarker discovery.
Native antigen fractionation protein microarrays for biomarker discovery.
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用于生物标志物发现的天然抗原分级蛋白质微阵列。
DOI：
10.1007/978-1-61779-043-0_9
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Liu,BrianC-S
中科院分区：
文献类型：
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作者：
CaiazzoJr,RobertJ;O'Rourke,DennisJ;Barder,TimothyJ;Nelson,BryceP;Liu,BrianC-S
在该方案中，我们使用T24人膀胱癌细胞系作为天然抗原源来构建分级裂解微阵列。随后，这些芯片被用来比较间质性膀胱炎/痛性膀胱综合征(IC/PBS)患者和正常女性对照组的自身抗体反应。为了做到这一点，T24细胞在非变性条件下裂解以获得天然抗原。然后用PF-2D液相色谱对这些天然抗原进行2D分离；第一维通过等电点分离蛋白质，第二维根据疏水性分离蛋白质。将得到的蛋白质组分打印到涂有硝基纤维素的玻璃片(路径载玻片)上，以创建一组分级裂解物微阵列。为了比较IC/PBS患者和正常对照的自身抗体反应，将分离的裂解物阵列与从IC/PBS患者和对照血清中纯化的荧光标记的IgG样本进行竞争性杂交。该方案详细描述了用于自身抗体分析的本地抗原分级裂解物微阵列的创建和使用。
In this protocol, we used the T24 human bladder cancer cell line as a source of native antigens to construct fractionated lysate microarrays. Subsequently, these microarrays were used to compare the autoantibody responses of individuals with interstitial cystitis/painful bladder syndrome (IC/PBS) to those of normal female controls. To accomplish this, T24 cells were lysed under nondenaturing conditions to obtain native antigens. These native antigens were then fractionated in 2D using a PF-2D liquid chromatography; the first dimension separated the proteins by their isoelectric points, and the second separated them according to hydrophobicity. The resulting protein fractions were printed onto nitrocellulose-coated glass slides (PATH slides) to create a set of fractionated lysate microarrays. To compare the autoantibody responses of IC/PBS patients with normal controls, the fractionated lysate arrays were competitively hybridized with fluorescently labeled IgG samples purified from both IC/PBS and control sera. This protocol presents a detailed description of the creation and use of native antigen fractionated lysate microarrays for autoantibody profiling.