THE MEASUREMENT OF INSOLUBLE PROTEINS USING A MODIFIED BRADFORD ASSAY
THE MEASUREMENT OF INSOLUBLE PROTEINS USING A MODIFIED BRADFORD ASSAY
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DOI：
10.1016/0003-2697(88)90199-6
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发表时间：
1988-09-01
影响因子：
2.9
通讯作者：
PATERSON, WR
中科院分区：
文献类型：
--
作者：
GOTHAM, SM;FRYER, PJ;PATERSON, WR
描述了用于测定热变性的不溶性蛋白质的量的技术。已经使用四种球状蛋白质、牛血清白蛋白、β-葡聚糖、葡聚糖和葡聚糖来验证该测定法。乳球蛋白、溶菌酶和卵清蛋白。它包括使用8 M尿素和5% 2-巯基乙醇的再溶解方案，与Bradford染料结合试验相关。在100 ℃下进行再溶解方案以能够完全回收所有不溶性蛋白质。β-乳球蛋白再溶解在加热1分钟后完成，而牛血清白蛋白、溶菌酶和卵清蛋白的样品需要加热1.5分钟。该测定法可以测量小至10 μ g的蛋白质浓度，通常标准偏差为3%，因此与标准Bradford测定法相比是有利的。如果不干扰Bradford试验，则可采用该技术研究其他类型的变性，例如导致后续不溶性的化学变性。
A technique for determining the amount of thermally denatured, insoluble protein is described. The assay has been validated using four globular proteins, bovine serum albumin, .beta.-lactoglobulin, lysozyme, and ovalbumin. It consists of a resolubilization protocol, using 8 M urea and 5% 2-mercaptoethanol, linked to the Bradford dye binding assay. The resolubilization protocol was carried out at 100.degree.C to enable complete recovery of all insoluble proteins. .beta.-Lactoglobulin resolubilization was completed after heating for 1 min, whereas samples of bovine serum albumin, lysozyme, and ovalbumin required heating for 1.5 min. The assay can measure protein concentrations as small as 10 .mu.g, typically with standard deviations of 3%, thus comparing favorably with the standard Bradford assay. Other types of denaturation, such as chemical denaturation causing subsequent insolubility, may be studied with this technique providing that there is no interference with the Bradford assay.