小中大4. The BCA assay will produce a linear response over a wide concentration range; however, to extend the dynamic range of the data analysis a quadratic response can be used to model the data.
BCA分析在宽浓度范围内有着线性结果；但是扩展数据分析的动力学范围，可以应用二次方程来拟合数据。
7.3. Comments
A microbased BCA assay can be used to improve the sensitivity of the procedure (1–25 ug/ml). The microbased assay uses a more concentrated working solution and can be prone to precipitation; again commercial sources of this modified BCA assay are available (Pierce). The BCA assay is sensitive to either copper chelators (e.g., EDTA) or reagents that can also reduce Cu2t (e.g., DTT), a summary of the maximum tolerances can be found in Table 8.1.
评价
微量BCA法可以用来提高分析的灵敏度（1-25ug/ml）。微量BCA法使用更高浓度的工作液，更易于沉淀；这种改进型的BCA法有商品化的来源（Pierce）。BCA法对铜离子螯合剂（如EDTA）敏感，对其它能够还原二价铜离子的试剂敏感，表8.1总结了BCA法对这些干扰的最大耐受性。
8. Amine Derivatization (Range: 0.05–25 ug)
氨基衍生法
（略）
9. Detergent-Based Fluorescent Detection(Range: 0.02–2 ug)
表面活性剂荧光检测法
（略）
10. General Instructions
The choice of measurement format used will depend on the throughput, sensitivity, and precision required of the assay, and concerns about assay interferences that can be reduced by dilution in cuvette-based assays. From our experience, both in industry and academia, plate-based assays are replacing cuvette assays due to increases in throughput. For all spectrophotometric techniques, the instrument should be warmed up for 15 min prior to measurement and any calibration programs run before sample analysis. Samples and reagents should be equilibrated to room temperature before analysis to avoid condensation on optical surfaces.
一般通则
检测模式的选择依赖于检测通量、灵敏度和分析所要求的精密度，以及对检测干扰物质的考虑。干扰物质在比色皿分析中可以用稀释来降低。从我们工业和学术两方面的经验来看，由于检测通量的提高，板式法正在取代比色皿法。所有分光光度技术的要求，仪器在测量前要预热15min，在样品分析前运行好所有的校正程序。为了避免光学表面的冷凝水，分析前样品和试剂都应当平衡至室温。
10.1. Cuvettes
Traditionally, cuvettes have been used for the majority of spectrophotometric protein assays. Quartz cuvettes can be costly, therefore glass cuvettes are preferred; however, both of these may have to be washed between measurements to remove dye and adsorbed protein. Disposable plastic cuvettes are available and can be used to increase the throughput where many samples have to be measured, or the reagent is prone to sticking to the cuvette surface, for example Bradford reagent. Staggering of sample analysis is especially important if the signal is not stable, or does not run to completion within the time frame of the assay, for example BCA or Lowry assays. The best precision is obtained from a two-beam instrument incorporating a reference cell to account for instrument drift. Replacing the cuvette in the holder between each measurement due to cleaning, or the use of disposable cuvettes can result in changes in alignment, resulting in significant changes in amount of light reaching the detector. This is especially important if low volume cuvettes are being used where the transmission window is reduced in size. Care should also be taken with low-volume cuvettes to ensure the sample covers the entire transmission window.
比色皿
传统上，比色皿用于分光光度法的蛋白质分析。石英比色皿较贵，因而玻璃比色皿用的更多；但是，两种比色皿在使用当中都要清洗，以去除染料和吸附的蛋白。一次性的塑料比色皿可以用来提高需检测样品多时的检测通量，或试剂易于粘附到比色皿表面时，例如Bradford试剂。如果检测信号不稳定，或在检测的时间窗口内不能完成操作，样品的交叉排列分析就特别重要，例如BCA法和Lowry法。用配备参比池的双光束仪器来补偿仪器的漂移，可以得到最佳的准确度。由于清洁或使用一次性比色皿，在每次测量间更换比色架中的比色皿，会导致光直线度方面的变化，使得到达检测器的光量发生重大变化。在使用小体积的比色皿时，透射窗口的大小会减少，这一点尤其重要。在使用小体积比色皿时，还要小心保证样品覆盖了整个透射窗口。
Care should be taken when handling and cleaning cuvettes. Prevent fingerprints from contaminating the transmitting surfaces. Cuvettes should be washed with either water or an appropriate solvent between runs and dried using a stream of nitrogen gas. If smearing of the transmitting surface is observed, the cuvette can be rewashed in water, ethanol, and finally acetone, or removed using ethanol and lintless lens tissue. If protein deposition is a recurring problem, cuvettes can be washed overnight in nitric acid and thoroughly washed before use.
在操作和清洁比色皿时要小心。防止指纹污染透射表面。在每次操作中间，要用水或其他适当的溶剂清洗比色皿，氮气吹干。如果比色皿透射面有污点，可以用水、乙醇，最后用丙酮再次清洗，或者用乙醇和无纺的镜头纸除去。如果经常发生蛋白质沉淀问题，比色皿要在硝酸中浸洗过夜，再次使用前彻底清洗。
10.2. Microwell plates
The majority of protein assays have been adapted for use in microwell plates, typically 96-well plates to enhance speed, throughput and lower sample and reagent usage. Many of the commercial fluorescent assays are specifically designed for plate formats. The plate reader format also offers the advantage of being able to read multiple samples within a short period (typically 25 s) reducing potential timing differences in reactions that do not go to completion, or are unstable.
大部分蛋白质分析方法都可以调整后使用微孔板，典型的如96孔板，以加快速度、提高通量、减少样品和试剂的使用量。很多商品化的荧光分析方法特别设计来用板式模式。板式仪器模式也有短时间内（通常25s）能够读出多个样品值的优势，可以减小不完全反应或不稳定反应中潜在的时程差异。
Protein UV measurements can be made in a plate format; however, the effective path length can be difficult to calculate due to meniscus formation for concentrated protein solutions (many commercial plate readers can estimate effective path-length and thereby improving protein quantitation calculations). Quartz 96-well plates tend to be expensive, difficult to clean and prone to scratches that can affect light transmission.
蛋白质的紫外检测也可以用板式模式；但是，由于浓蛋白溶液中弯曲液面的形成，使得难以计算有效的光路长度（很多商品化的板式仪器能够估计有效光路长度，因而可以改善蛋白质的定量计算）。石英材质的96孔板太昂贵了，也难于清洗、易于划伤而影响光透射。
Care should be taken in the preparation of protein assays in plate formats. The use of lower volume samples (down to 5 ul for some assays) can increase the relative pipetting errors of high viscosity solutions. Well-to-well contamination should be avoided by using fresh pipette tips for each sample and reagent. Regular calibration of the instrument should be performed using either optical standards or solid phase fluorescent standard plates (Matech) to ensure equal transmission/light detection from all wells. Many of the 96-well plates conform to a standard geometry; however, in our experience, it is worth analyzing plate geometry in the plate-reader, especially if a different plate supplier is used to ensure equal illumination, and detection for fluorescent-based measurements. Plate-based assays can also be more sensitive to sample precipitation (common in the Lowry and Bradford assays) when compared to cuvette-based assay due to the detection geometry.
Recently, spectrophotometers that can measure low microliter samples (typically 1–2 ul), without the need for a cuvette or microplates have become commercially available (Tecan and Thermo Scientific), further minimizing sample usage.
