DETERMINATION OF MICROGRAM QUANTITIES OF PROTEIN IN THE PRESENCE OF MILLIGRAM LEVELS OF LIPID WITH AMIDO BLACK B-10
DETERMINATION OF MICROGRAM QUANTITIES OF PROTEIN IN THE PRESENCE OF MILLIGRAM LEVELS OF LIPID WITH AMIDO BLACK B-10
复制标题
DOI：
10.1016/0003-2697(85)90445-2
复制
发表时间：
1985-01-01
影响因子：
2.9
通讯作者：
PEDERSEN, PL
中科院分区：
文献类型：
--
作者：
KAPLAN, RS;PEDERSEN, PL
已经开发出一种能够在存在非常高水平的脂质(即20-40毫克)的情况下准确地测定低含量蛋白质(即2-24微克)的方法。该方法是在Schaffner和Weissmann(W.Schaffner和C.Weissmann，1973，肛门)最初的Amido Black 10B蛋白分析的基础上发展起来的。生物化学。56,502-514)，并结合了几个关键的修改，使该分析能够在不受干扰的情况下对含脂样品进行分析。这些修改包括大幅增加分析体积(从而降低最终的脂类浓度)以及十二烷基硫酸钠和三氯乙酸的浓度。在此条件下，在无脂和有脂(20 Mg)存在下，牛血清白蛋白的量均为2-24微克，呈线性标准曲线。此外，原始样品中高达40毫克的脂肪不会对该分析产生影响。用线粒体蛋白质(即，亲水和疏水蛋白质的混合物)的样本也证明了线性和不受脂质(20毫克)的干扰。此外，我们还表明，在蛋白质(20微克)和脂质(20毫克)存在的情况下，高浓度的各种缓冲液、盐和非离子洗涤剂不会干扰测定。最后，与现有的几种蛋白质估计方法相比，这种新的方法在不受干扰的情况下提高了容忍高脂水平的能力。该方法可广泛应用于含蛋白脂质体的重组体系中的蛋白质测量。这类研究通常需要在洗涤剂存在的情况下和在存在非常高的脂/蛋白比的条件下将少量蛋白质加入脂质体中。据我们所知，这是第一次证明在存在如此高浓度的脂质的情况下，分光光度分析能够准确地测量低含量的蛋白质。
A method which is capable of accurately determining low amounts of protein (i.e., 2-24 .mu.g) in the presence of very high levels of lipid (i.e., 20-40 mg) has been developed. The procedure was developed from the original amido black 10B protein assay of Schaffner and Weissmann (W. Schaffner and C. Weissmann, 1973, Anal. Biochem. 56, 502-514) and incorporates several critical modifications that enable the assay to be performed with lipid-containing samples without interference. The modifications include a substantial increase in the assay volume (thereby decreasing the final lipid concentration) as well as the sodium dodecyl sulfate and trichloroacetic acid concentrations. Under these conditions, a linear standard curve is obtained with 2-24 .mu.g of bovine serum albumin in both the absence and the presence of lipid (20 mg). Moreover, the assay is unaffected by as much as 40 mg of lipid in the original sample. Linearity as well as noninterference by lipid (20 mg) is also demonstrated with a sample of mitochondrial protein (i.e., a mixture of hydrophilic and hydrophobic proteins). Additionally, we show that in the presence of protein (20 .mu.g) and lipid (20 mg), high concentrations of various buffers, salts, and nonionic detergents do not interfere with the assay. Finally, the enhanced ability of this new method to tolerate high lipid levels without interference relative to several existing protein estimation methods is demonstrated. This procedure should prove widely useful for measuring protein in reconstituted systems involving proteoliposomes. Such studies often require the incorporation of small amounts of protein into liposomes in the presence of detergent and under conditions where a very high lipid/protein ratio exists. To our knowledge, this is the first demonstration that a spectrophotometric assay is capable of accurately measuring low amounts of protein in the presence of such high concentrations of lipid.