Sheets Rachelle, Rajaboina Bhavik, Bromberg Caitlin E, Curtin Liam P, Haddock Mitchell L, Stafford Phillip, Thomas Theresa Currier, Scheck Adrienne C
Department of Child Health, University of Arizona College of Medicine-Phoenix, Phoenix, AZ, USA.
Department of Translational Neurosciences, University of Arizona College of Medicine-Phoenix, Phoenix, AZ, USA.
Biotechniques. 2025 Mar;77(3):95-102. doi: 10.1080/07366205.2025.2502268. Epub 2025 May 23.
Total protein isolation followed by quantitation using a colorimetric method, such as the bicinchoninic acid (BCA) assay is a common laboratory protocol. Protein concentrations are determined by comparing extracted samples to a standard curve generated from serial dilutions of a reference protein, such as bovine serum albumin (BSA). This study aimed to identify the most reproducible and accurate method for quantifying protein concentrations in an experimental series over time. We analyzed the effect of serial freeze-thaws, inter-person and intra-person variability in standard preparation and assay execution. Absorbance was measured at 565 nanometers (nm) using an Epoch Microplate Spectrophotometer (Agilent Technologies, Inc., Santa Clara, CA) with Gen5 Data Analysis software. The most consistent and accurate method for determining the protein concentrations over time is to prepare a large batch of diluted BSA standards, aliquot them into small portions, and store them frozen.
采用比色法（如二辛可宁酸（BCA）测定法）进行总蛋白分离并随后进行定量是一种常见的实验室方案。通过将提取的样品与由参考蛋白（如牛血清白蛋白（BSA））的系列稀释液生成的标准曲线进行比较来确定蛋白质浓度。本研究旨在确定在一系列实验中随时间推移定量蛋白质浓度的最可重复和准确的方法。我们分析了系列冻融、标准品制备和测定执行过程中的人际和个人内变异性。使用配备Gen5数据分析软件的Epoch微孔板分光光度计（安捷伦科技公司，加利福尼亚州圣克拉拉）在565纳米（nm）处测量吸光度。随时间推移测定蛋白质浓度最一致和准确的方法是制备一大批稀释的BSA标准品，将它们分成小份，然后冷冻保存。
