UV measurements in microplates suitable for high-throughput protein determination
UV measurements in microplates suitable for high-throughput protein determination
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DOI：
10.1016/s0003-2697(02)00460-8
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发表时间：
2003-02-15
影响因子：
2.9
通讯作者：
Horn, A
中科院分区：
文献类型：
--
作者：
Kreusch, S;Schwedler, S;Horn, A
本文介绍了一种用微板紫外分光光度测定蛋白质的方法。使用spectraMax plus阅读器、UVStar 96和384孔微板以及96或384平行通道液体处理技术，可以在1至8000杯蛋白质/毫升的范围内进行大规模测定，精密度优于3%的变异系数(CV)，测量波长为205、215和280 nm，并使用不同的体积相关光路长度。由于只有阅读器的路径检查选项，八种不同蛋白质在205 nm处的吸光度系数为30ml/(Mg X Cm)，变异系数为5.6%，因此无需任何单独的校准即可确定蛋白质浓度。分析体积范围为60-250微升的样品，不需要耗时和昂贵的处理，也不会造成样品损失。使用特殊的96或384平行透析装置，可以有效地去除因紫外线吸收而干扰分析的低分子物质，如缓冲液和洗涤剂。在强紫外线吸收洗涤剂Triton X-100存在的情况下，也显示了血清蛋白质分离的应用实例。(C)2003年埃尔塞维尔科学公司(美国)。版权所有。
An UV spectrophotometric method for protein determination using microplates is described. Using the SPECTRAmax PLUS reader, the UVStar 96- and 384-well microplates and a 96 or 384 parallel channel liquid handling technique, large-scale determinations can be performed with intraassay precision better than 3% CV (coefficient of variation) in the range from 1 to 8000 mug of protein/ml, measuring at 205, 215, and 280 nm and using different volume-dependent light-path lengths. Since the absorbance coefficient at 205 nm is found to be 30 ml/(mg x cm) for eight different proteins with a CV of 5.6% only with the Path Check option of the reader, protein concentration can be determined without any individual calibration. Samples in the volume range of 60-250 mul can be analyzed without time-consuming and expensive treatment and without sample loss. Using a special 96 or 384 parallel dialyzing device, low molecular weight substances which interfere with the analysis by their UV absorbance, such as buffers and detergents, can effectively be removed. Application examples for serum protein separation are also shown in the presence of the strongly UV absorbing detergent Triton X-100. (C) 2003 Elsevier Science (USA). All rights reserved.