Absolute quantitation of endogenous proteins with precision and accuracy using a capillary Western system.
Absolute quantitation of endogenous proteins with precision and accuracy using a capillary Western system.
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DOI：
10.1016/j.ab.2013.07.022
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发表时间：
2013-11-01
影响因子：
2.9
通讯作者：
Goldsmith PK
中科院分区：
文献类型：
--
作者：
Chen JQ;Heldman MR;Herrmann MA;Kedei N;Woo W;Blumberg PM;Goldsmith PK
在复杂的生物环境中精确和准确地定量蛋白质表达水平是具有挑战性的。在这里，我们描述了一种使用自动化毛细管免疫测定系统（基于大小的简单蛋白质系统，ProteinSimple，CA）绝对定量细胞裂解物中内源性蛋白质的方法。该方法能够准确地测量每纳克细胞裂解物中皮克或亚皮克水平的靶蛋白的绝对量。该测量不依赖于细胞基质或细胞裂解缓冲液，并且不受不同抗体对其特异性表位的亲和力的影响。然后，我们应用这种方法来定量蛋白激酶C（PKC）亚型在LNCaP和U937细胞，两个细胞系广泛用于探测下游的生物反应PKC靶向配体的表达的绝对水平。我们的绝对定量证实了PKCδ在两种细胞中的优势，支持这种PKC亚型在这些细胞系中的重要功能作用。本文所述的方法提供了一种准确定量蛋白质表达水平并将蛋白质水平与功能相关联的方法。除了相对于常规蛋白质印迹分析提高的准确性之外，它还避免了在比较来自具有不同亲和力的不同抗体的信号强度时固有的失真。
Precise and accurate quantification of protein expression levels in a complex biological setting is challenging. Here, we describe a method for absolute quantitation of endogenous proteins in cell lysates using an automated capillary immunoassay system (the size-based Simple Western system, ProteinSimple, CA). The method was able to accurately measure the absolute amounts of target proteins at picogram or sub-picogram levels per nanogram of cell lysates. The measurements were independent of the cell matrix or the cell lysis buffer and were not affected by different antibody affinities for their specific epitopes. We then applied this method to quantitate absolute levels of expression of protein kinase C (PKC) isoforms in LNCaP and U937 cells, two cell lines used extensively for probing the downstream biological responses to PKC targeted ligands. Our absolute quantitation confirmed the predominance of PKCδ in both cells, supporting the important functional role of this PKC isoform in these cell lines. The method described here provides an approach to accurately quantitate levels of protein expression and correlate protein level with function. In addition to enhanced accuracy relative to conventional western analysis, it circumvents the distortions inherent in comparison of signal intensities from different antibodies with different affinities.
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影响因子：
9.3
作者：
Ellington AA;Kullo IJ;Bailey KR;Klee GG
通讯作者：
Klee GG
影响因子：
48
作者：
Gillette, Michael A.;Carr, Steven A.
通讯作者：
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影响因子：
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作者：
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通讯作者：
Kazanietz, Marcelo G.
影响因子：
2.9
作者：
Yang-Boja, E;DeFilippes, F;Fales, HM
通讯作者：
Fales, HM
影响因子：
--
作者：
de Sousa Abreu R;Penalva LO;Marcotte EM;Vogel C
通讯作者：
Vogel C