Application Note
Protein quantitation with the EMax Plus Microplate Reader
- 8 filters come standard to cover a wide range of applications
- Compact footprint
- Walk-up usability
- Pre-defined protocols with SoftMax Pro Software
Introduction
Endpoint readers are prolific in the laboratory since absorbance has become the detection of choice for many applications. Examples include ELISAs for quantitation of cytokines and protein concentration determination using the Bradford assay. This application note compares the performance of the Molecular Devices EMax® Plus Microplate Reader to the VMax Reader using a Bradford protein quantitation assay. In addition, a performance comparison is made between the EMax Plus Microplate Reader and the EMax Endpoint Reader using a sandwich ELISA for the quantitation of mouse/rat IL-22.
Bradford assay
The Bradford protein concentration assay is an absorbance assay based upon the addition of an acidic dye to a protein solution1 . The absorbance maximum for Coomassie Blue dye shifts from 460 nm to 595 nm when binding to protein occurs2.Comparing unknown samples to a standard curve provides a relative measurement of protein concentration.
The Bio-Rad Bradford Protein Assay Kit was used for quantitation. A standard curve of bovine serum albumin in the sensitivity range of the assay was prepared in triplicate and pipetted into a 96-well microplate. Coomassie Brilliant Blue G-250 dye was diluted 1:4 per instructions and added to the protein samples. After a 30 minute incubation period, the optical density was read at 585 nm on both the EMax Plus and the VMax readers. SoftMax® Pro Software was used to subtract the plate blank and generate a standard curve (Figure 1). Comparison of data from both instruments showed that the R2 value for both curves was 0.98. The linear portion of the curve was comparable to the curve in the kit literature.
Figure 1: Absorbance reader comparison with Bradford protein assay. The Bio-Rad Bradford Protein Assay Kit was used to determine the concentrations of bovine serum albumin in a standard curve. Signal comparison from both instruments was nearly identical. The assay maintained linearity across five dilutions with R2 values = 0.98.
ELISA assay
Interleukin-22 is a cytokine involved in immune function. It is part of the Interleukin-10 superfamily of cytokines and the family of interferons3 and it is an important mediator in inflammatory response and tissue repair. IL-22 is expressed on many cells including gut, skin, NK cells and CD4+ Th1 cells4 . A quantitative mouse/rat IL-22 sandwich ELISA from R&D Systems was used to compare results from EMax Plus and EMax microplate readers. A polyclonal antibody specific to mouse/rat IL-22 was pre-coated onto a 96-well microplate. Standards, controls and blanks were pipetted into the wells and incubated followed by washing with the Molecular Devices MultiWash™+ Microplate Washer. An enzyme-linked polyclonal antibody specific for mouse/rat IL-22 was added to the wells and incubated followed by an additional wash. Substrate was added followed by stop solution after another short incubation. Absorbance was read on both the EMax Plus and the EMax microplate readers. Color intensity was proportional to the amount of mouse or rat IL-22 bound during the initial incubation. SoftMax Pro Software was used to calculate each standard curve shown in Figure 2. R2 values for each curve were also very close.
Figure 2: Absorbance reader comparison with ELISA assay. A Mouse/Rat IL-22 Quantikine ELISA from R&D Systems was used to compare performance of the EMax Plus and the EMax endpoint readers in an absorbance assay. An IL-22 standard curve was prepared and a sandwich ELISA performed using the MultiWash+ Plate Washer in strip mode to wash the wells. After reading the ELISA plate on both readers, each standard curve was nearly identical.
MultiWash+ Microplate Washer
The MultiWash+ Microplate Washer is an automated washer that is configurable for both 96- and 384- well plates. It comes standard with 4 wash/rinse bottles and 1 waste bottle for maximum washing flexibility. The on-board touch panel comes with the option of 20 different wash protocols with up to 8 cycles within each protocol. Washing variations include adjustable speed and volume, adjustable aspiration speed and time and adjustable soak times. Efficient washing is achieved with cross-wise aspiration reducing residual volume within each well. Three modes of shaking are available to mix solutions. The washer has a small footprint and is vacuum and pressure free, with on-board pumps that provide a quiet wash experience.
3-step process for setup and data analysis
Step 1. SoftMax Pro assay setup. The Plate Setup Wizard walks the user through the steps to set up the assay. The software automatically recognizes instruments that are connected. It then uses the pre-defined settings for the specified instrument to build a protocol. It this case, the EMax Plus Microplate Reader was the default.
Step 2. Plate layout configuration. User selects appropriate wells for samples and control using plate configuration tool.
Step 3. Data acquisition and analysis. Predefined settings for Bradford assays are used to automatically calculate graphs from plate data.
Summary
The EMax Plus Microplate Reader provides a robust solution in an entry level platform. The convenience and power of SoftMax Pro Software makes it possible to easily set up and analyze a broad range of assays. Results from both a protein quantitation assay and an ELISA assay have shown performance equivalent to high end microplate readers.
References
- Bradford, M. (1976) Anal Biochem. 72:248
- Sedmack, J et al. (1977) Anal Biochem. 79:544
- Pesta, S. et al. (2004) Annual Rev Immunol. 22:929-79
- Liang, S.C. et al. (2005) J. Exp. Med. 203:2271
介绍
随着光吸收法可以满足越来越多的应用检测 领域，终点法读板机在实验室中的占有率也 越来越高。例如ELISAs方法进行细胞因子 定量和Bradford法进行蛋白定量。本篇应用 就是要着重比较使用Bradford法进行蛋白定 量检测时，Molecular Devices公司的产品 EMax® Plus微孔板读板机和VMax微孔板读 板机的性能。此外，我们比较了Emax Plus 微孔板读板机和Emax终点法微孔板读板 机，利用双抗夹心ELISA方法来定量小鼠/大 鼠IL-22细胞因子时表现上的差异。
Bradford 实验
Bradford蛋白定量检测法是一种基于酸性染料与蛋白结合的光吸收实验1。当考马斯亮蓝染料与蛋白质结合后，其光吸收最大检测波峰是从460nm转移至595nm2。可以通过拟合标准曲线得出未知样本的浓度。
使用伯乐公司的Bradford蛋白定量检测试剂 盒。以牛血清白蛋白标品来制作一条标准曲 线，加入96孔板中且每个浓度下有三个复 孔。将考马斯亮蓝G-250染料按1:4稀释后 加入到蛋白样本中。孵育30分钟后，分别在 EMax Plus和VMax两台读板机上检测585nm 波长下的吸光度。最后，应用SoftMax® Pro 软件扣除背景后并生成一条标准曲线(图1)。 通过比较发现两台读板机上得出的R2结果都 是0.98。曲线的线性部分也和试剂盒中对标 准曲线的描述相同。
图 1. 使用伯乐公司的Bradford蛋白定量检测试剂盒中的牛血清白蛋白标准品制备一条标 准曲线。 两台仪器获得的结果比较几乎相同。 实验结果线性区间可达5个稀释度，且R2 结果均为0.98。p>
ELISA 实验
白介素22是一种和炎症相关的细胞 因子。它是白介素10细胞因子超家 族和干扰素3家族的一员，在介导炎 症反应和组织修复过程中起到重要的 作用。IL-22可表达在多种细胞中， 如gut，skin，NK细胞and CD4+ Th1细胞4。
使用R&D公司小鼠/大鼠IL-22双抗 夹心法ELISA检测试剂盒进行定 量，比较EMax Plus和EMax两台读 板机的检测结果。首先，将一种能特 异性结合小鼠/大鼠IL-22的多克隆抗 体预先包被在一块96孔板中。然 后，使用移液器向微孔板中加入标准 品、质控和空白对照进行孵育，推荐 使用Molecular Devices公司的洗板 机MultiWash™+进行洗板。随后， 包被有多抗的微孔板中加入小鼠/大 鼠IL-22细胞因子进行孵育后再洗 板。最后，短暂孵育后加入底物终止 反应并在EMax Plus和EMax两台读 板机上进行读数。颜色深浅与孵育时 结合的小鼠／大鼠IL-22量成正比。 应用SoftMax Pro软件计算后生成标 准曲线(图2)。两条标准曲线的R2值 也很接近。
图2. 使用R&D公司的小鼠/大鼠IL-22 ELISA定量检测试剂盒，比较EMax Plus和EMax两台读板机在光吸法检测结果上的差异。标准曲线的制备及双 抗夹心ELISA实验过程都使用了MultiWash+洗板机单列清洗模式。发现在 两台读板机上的ELISA检测结果，即两条标准曲线几乎相同。
About the MultiWash+ 洗板机
MultiWash+是一台全自动洗板机，兼容96孔和384孔板。并且标配有四个洗液瓶或冲洗瓶，外加一个废液瓶，实现更大的清洗灵活性。仪器上配有触摸屏并且预存20种不同的清洗模式，每种模式下均可灵活的设置8个循环步骤。另外，仪器还可调节洗液流速和体积，调节吸液的速度和时间及浸泡时间。
MultiWash+是一台全自动洗板机，兼容96孔和384孔板。并且标配有四个洗液瓶或冲洗瓶，外加一个废液瓶，实现更大的清洗灵活性。仪器上配有触摸屏并且预存20种不同的清洗模式，每种模式下均可灵活的设置8个循环步骤。另外，仪器还可调节洗液流速和体积，调节吸液的速度和时间及浸泡时间。
同时，洗头通过在每个孔中交叉吸液方式提高清洗效率，降低每孔中的残液量，并提供三种震荡方式方便用户混匀洗液。仪器占地面积小，无需额外的真空和压力装置，内置泵的设计为您提供一个安静的操作环境。
3-step process for setup and data analysis
Step 1. SoftMax Pro 实验设置 实验设置模块可指导用户一步一步的设置实验。软件会自动识别并连接仪器。然后软件会针对特定的读板机应用预设并建立模板。针对本次设置，EMaxPlus已被默认选择。
Step 2. 板布局设置 用户可利用设置工具选择对应的样品和质控品孔。
Step 3. 用户可利用设置工具选择对应的样品和质控品孔。 Bradford实验的的预设参数可以自动根 据读板的数据自动拟合出曲线。
总结
EMax Plus微孔板读板机可作为一个 入门级的自动化检测平台。方便、强 大的SoftMax Pro软件使得快速设置 与分析多种实验成为可能。通过蛋白 定量和ELISA的检测实验，我们发 现EMax Plus微孔板读板机表现出的 优异性能可与很多高端型读板机相媲 美。p>
文献
- Bradford, M. (1976) Anal Biochem. 72:248
- Sedmack, J et al. (1977) Anal Biochem. 79:544
- Pesta, S. et al. (2004) Annual Rev Immunol. 22:929-79
- Liang, S.C. et al. (2005) J. Exp. Med. 203:2271