Spectrophotometric determination of protein concentration.
Spectrophotometric determination of protein concentration.
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DOI：
10.1002/0471140864.ps0301s33
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发表时间：
2004-11-01
影响因子：
--
通讯作者：
Pace, C Nick
中科院分区：
文献类型：
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作者：
Grimsley, Gerald R;Pace, C Nick
溶液中纯化蛋白质的浓度最方便和准确的测量使用吸光度法。根据比尔-朗伯定律，吸光度A是摩尔浓度C的线性函数：A = λ x l x c，其中e是摩尔吸光系数，l是池路径长度。该单元提供了计算折叠或未折叠蛋白质的Δ的方案，利用蛋白质中三个贡献生色团（Trp，Tyr和Cys的侧链）的平均Δ值。一个基本的协议描述了如何使用计算的蛋白质浓度和比尔-朗伯定律。提供了一种灵敏的方法来测量含有很少的色氨酸或酪氨酸残基的蛋白质的浓度，并提供了一种简单的方法来估计粗提取物中的总蛋白质浓度。
The concentration of a purified protein in solution is most conveniently and accurately measured using absorbance spectroscopy. The absorbance, A, is a linear function of the molar concentration, C, according to the Beer-Lambert law: A = epsilon x l x c, where e is the molar absorption coefficient and l is the cell path length. This unit provides protocols for calculation of epsilon for a folded or unfolded protein, making use of the average epsilon values for the three contributing chromophores in proteins (the side chains of Trp, Tyr, and Cys). A basic protocol describes how to measure the concentration of a protein using the calculated epsilon and the Beer-Lambert law. A sensitive method is provided for measuring the concentration of proteins that contain few if any tryptophan or tyrosine residues, and a simple method is provided for estimating total protein concentration in crude extracts.