斑点印迹法定量检测蛋白质羰基化。
Quantitation of protein carbonylation by dot blot.
机构信息
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Bethesda, MD 20892, USA.
出版信息
Anal Biochem. 2012 Apr 15;423(2):241-5. doi: 10.1016/j.ab.2012.01.031. Epub 2012 Feb 8.
Abstract
Protein carbonylation is the most commonly used measure of oxidative modification of proteins. It is frequently measured spectrophotometrically or immunochemically by derivatizing proteins with the classical carbonyl reagent, 2,4-dinitrophenylhydrazine. We developed an immunochemical dot blot method for quantitation of protein carbonylation in homogenates or purified proteins. Dimethyl sulfoxide was employed as the solvent because it very efficiently extracts proteins from tissues and keeps them soluble. It also readily dissolves 2,4-dinitrophenylhydrazine and wets polyvinylidene difluoride (PVDF) membranes. The detection limit is 0.19 ± 0.04 pmol of carbonyl, and 60 ng of protein is sufficient to measure protein carbonyl content. This level of sensitivity allowed measurement of protein carbonylation in individual Drosophila.
摘要
蛋白质羰基化是最常用的蛋白质氧化修饰的衡量标准。它通常通过将经典羰基试剂 2,4-二硝基苯肼与蛋白质衍生化来分光光度法或免疫化学法测量。我们开发了一种免疫化学点印迹法来定量测定匀浆或纯化蛋白质中的蛋白质羰基化。二甲基亚砜被用作溶剂，因为它非常有效地从组织中提取蛋白质并保持其可溶性。它还容易溶解 2,4-二硝基苯肼并润湿聚偏二氟乙烯 (PVDF) 膜。检测限为 0.19 ± 0.04 pmol 羰基，60 ng 蛋白质足以测量蛋白质羰基含量。这种灵敏度水平允许测量单个果蝇中的蛋白质羰基化。
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