CEA, IBEB, Lab Bioenerget Cellulaire, Saint-Paul-lez-Durance, F-13108, France.
J Chromatogr A. 2013 Mar 15;1281:135-41. doi: 10.1016/j.chroma.2013.01.061. Epub 2013 Jan 24.
The hydrophobic nature of membrane proteins (MPs) necessitates the use of detergents for their extraction, solubilization and purification. Because the concentration of amphiphiles is crucial in the crystallization process, detergent quantification is essential to routine analysis. Here we describe a quantitative high-performance thin-layer chromatography (HPTLC) method we developed for the detection of small quantities of detergent bound to solubilized MPs. After optimization of aqueous deposit conditions, we show that most detergents widely used in membrane protein crystallography display distinctive mobilities in a mixture of dichloromethane, methanol and acetic acid 32:7.6:0.4 (v/v/v). Migration and derivatization conditions were optimized with n-dodecyl-β-D-maltoside (DDM), the most popular detergent for membrane protein crystallization. A linear calibration curve very well fits our data from 0.1 to 1.6 μg of DDM in water with a limit of detection of 0.05 μg. This limit of detection is the best achieved to date for a routine detergent assay, being not modified by the addition of NaCl, commonly used in protein buffers. With these chromatographic conditions, no prior treatment is required to assess the quantities of detergent bound to purified MPs, thus enabling the quantification of close structure detergents via a single procedure. This HPTLC method, which is fast and requires low sample volume, is fully suitable for routine measurements.
膜蛋白（MPs）的疏水性需要使用去污剂来提取、溶解和纯化。因为在结晶过程中两亲物的浓度是至关重要的，所以去污剂的定量分析对于常规分析是必不可少的。在这里，我们描述了一种定量高效薄层层析（HPTLC）方法，我们开发了这种方法来检测溶解的 MPs 结合的少量去污剂。在优化水相沉积条件后，我们表明，在二氯甲烷、甲醇和乙酸 32:7.6:0.4（v/v/v）的混合物中，膜蛋白结晶中广泛使用的大多数去污剂都显示出独特的迁移率。用 n-十二基-β-D-麦芽糖苷（DDM）优化了迁移和衍生化条件，DDM 是用于膜蛋白结晶的最流行的去污剂。DDM 在水中的浓度从 0.1 到 1.6 μg 范围内的线性校准曲线拟合度非常好，检测限为 0.05 μg。这是迄今为止在常规去污剂分析中达到的最佳检测限，不受常用的蛋白质缓冲液中 NaCl 的添加影响。使用这些色谱条件，无需对纯化的 MPs 结合的去污剂进行预处理，因此可以通过单一程序对紧密结构的去污剂进行定量。这种 HPTLC 方法快速，所需样品体积小，完全适合常规测量。
