Optimization of a malachite green assay for detection of ATP hydrolysis by solubilized membrane proteins

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• 作者：Boris Repen^a ; Erwin Schneider^b ; Ulrike Alexiev^a ; ^{alexiev@physik.fu-berlin.de}
• 关键词：Inorganic phosphate detection ; Malachite green ; Integral membrane proteins ; Enzymatic ATP hydrolysis ; ABC transporter ; Fluorescent reporter groups
• 刊名：Analytical Biochemistry
• 出版年：2012
• 期刊代码：93_00032697
• 类别：imm
• 出版时间：15 July, 2012
• 卷：426
• 期：2
• 页码：103-105
• 文件大小：320 K

摘要

We studied the activity of the fluorescently labeled membrane transporter MalGFK₂, which transports maltose at the expense of ATP hydrolysis. We used a commercially available malachite green assay (SensoLyte MG phosphate assay kit; Anaspec) to quantify the liberated phosphate upon ATP hydrolysis. However, strong variations in phosphate concentration were measured when using the supplier鈥檚 handling protocol. We optimized the protocol, taking into account the effects mediated by glycerol, SDS, and fluorescent label on the sample. As a result we obtained highly reproducible phosphate concentration values under conditions optimal for solubilized membrane proteins.