A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts.
A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts.
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DOI：
10.1016/j.soilbio.2013.08.017
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发表时间：
2013-12
影响因子：
9.7
通讯作者：
Goulding, K. W. T.
中科院分区：
文献类型：
--
作者：
Redmile-Gordon, M. A.;Armenise, E.;White, R. P.;Hirsch, P. R.;Goulding, K. W. T.
关键词：
土壤萃取物通常含有大量溶解的腐殖化有机物，多酚类物质含量高。由于多酚严重混淆了提取蛋白质的定量，因此最小化这种干扰对于确保测量具有代表性非常重要。虽然布拉德福德比色法在土壤科学中经常用于快速定量土壤提取物中的蛋白质，但它有几个局限性。因此，我们研究了一种基于Lowry测定法的替代比色技术（通常用于测量微生物生物膜中作为不同池的蛋白质和腐殖质物质）。在因子组合中比较了Bradford法和改良的Lowry微孔板法的准确性。定量土壤提取物（用柠檬酸盐提取）中的蛋白质，包括标准添加模型蛋白（BSA）和多酚（Sigma H1675-2）。使用所描述的Lowry微孔板试验，即使浓度比通常用于提取土壤蛋白质的浓度高5倍，也没有检测到柠檬酸盐的干扰效应。此外，Bradford实验被发现极易受到两种同时发生且相互混淆的伪影的影响：1)由于添加的蛋白质而产生的显色受到多酚浓度的极大抑制；2)大量显色是由多酚的添加直接引起的。相比之下，Lowry方法能够区分蛋白质和非蛋白质来源的颜色形成，提供更准确的定量分析。这些结果表明，改进的lowry方法是一种更合适的提取物蛋白质（由标准当量定义）的测量方法，因为它较少受到土壤提取物中典型的高多酚含量的混淆。比较了Bradford和Lowry测量土壤提取物中蛋白质的技术。我们发现多酚含量对Bradford估计的影响远远大于Lowry。柠檬酸盐（通常用于提取土壤蛋白质）在Lowry试验中没有发现问题。采用Lowry微孔板法，对多酚和蛋白质的分析反应进行了分析。
Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts. Bradford and Lowry techniques for measuring protein in soil extracts were compared. We found polyphenol content affected Bradford estimations substantially more than Lowry. Citrate (often used to extract soil protein) was not found to be problematic for the Lowry assay. Using the Lowry microplate method, analytical response due to polyphenol and protein is ascribed.
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影响因子：
9.7
作者：
Bonmati, Manuel;Ceccanti, Brunello;Valero, Jordi
通讯作者：
Valero, Jordi
影响因子：
4.2
作者：
Emran, M.;Gispert, M.;Pardini, G.
通讯作者：
Pardini, G.
影响因子：
4.2
作者：
Giagnoni, L.;Magherini, F.;Renella, G.
通讯作者：
Renella, G.
影响因子：
4.4
作者：
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通讯作者：
FIRESTONE, MK
影响因子：
9.7
作者：
Gillespie, Adam W.;Farrell, Richard E.;Blyth, Robert I. R.
通讯作者：
Blyth, Robert I. R.