Protein array staining methods for undefined protein content, manufacturing quality control, and performance validation
Protein array staining methods for undefined protein content, manufacturing quality control, and performance validation
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DOI：
10.1016/j.ab.2006.08.035
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发表时间：
2006-12-01
影响因子：
2.9
通讯作者：
Chandler, Darrell P.
中科院分区：
文献类型：
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作者：
Schabacker, Daniel S.;Stefanovska, Ivana;Chandler, Darrell P.
评估由未定义蛋白质含量制备的蛋白质微阵列的质量和性能的方法需要阐明载玻片之间的可变性，并在相互作用测定后插入产生的信号强度值。因此，我们开发了几种简单的总修饰和翻译后修饰特异性芯片染色方法，以定量评估由二维液相分离（PF2D）技术衍生的全细胞裂解物体外蛋白组分制造的凝胶元件蛋白阵列的质量。观察到蛋白染色和固定蛋白含量的线性动态范围至少为3 log，深紫色蛋白染色和现场便携式微阵列成像仪的检测下限为每个凝胶元件8 pg蛋白。数据表明，结合PF2D和凝胶元件阵列方法，成功分离、转移和固定化了鼠疫耶尔森菌KIM D27的跨膜蛋白。内部牛血清白蛋白标准曲线提供了评估片上PF2D转移和定量每个凝胶元件固定的总蛋白的方法。因此，本文描述的基本PF2D阵列制造和质量保证/质量控制方法为开发用于询问宿主-病原体相互作用的全蛋白质组阵列提供了标准操作程序和基础，而不依赖于测序基因组、亲和标签或对靶细胞组成的先验知识。(c) 2006爱思唯尔公司版权所有。
Methods to assess the quality and performance of protein microarrays fabricated from undefined protein content are required to elucidate slide-to-slide variability and interpolate resulting signal intensity values after an interaction assay. We therefore developed several simple total- and posttranslational modification-specific, on-chip staining methods to quantitatively assess the quality of gel element protein arrays manufactured with whole-cell lysate in vitro protein fractions derived from two-dimensional liquid-phase fractionation (PF2D) technology. A linear dynamic range of at least 3 logs was observed for protein stains and immobilized protein content, with a lower limit of detection at 8 pg of protein per gel element with Deep Purple protein stain and a field-portable microarray imager. Data demonstrate the successful isolation, separation, transfer, and immobilization of putative transmembrane proteins from Yersinia pestis KIM D27 with the combined PF2D and gel element array method. Internal bovine serum albumin standard curves provided a method to assess on-chip PF2D transfer and quantify total protein immobilized per gel element. The basic PF2D array fabrication and quality assurance/quality control methods described here therefore provide a standard operating procedure and basis for developing whole-proteome arrays for interrogating host-pathogen interactions, independent of sequenced genomes, affinity tags, or a priori knowledge of target cell composition. (c) 2006 Elsevier Inc. All rights reserved.