automatically extracting the bradford assay data recorded with a smartphone

智能手机作为使用 Bradford 分析定量蛋白质的分析设备

Regardless of the downstream use (e.g., ELISA, enzyme kinetics, western blotting, protein purification, and mass spectrometry), protein quantification is crucial for accurate analysis in life sciences laboratories. In addition to their use as secondary readouts (i.e., to calculate relative levels of analytes per mass of protein), protein levels in a sample can also be the desired output itself. For example, one can be interested in protein levels in food resources1 or in urine2. There are many methods available to measure protein concentration in samples3, including direct UV absorbance readings4, protein-copper chelation5,6, protein-dye binding colorimetric assays7, and protein-dye binding fluorescent assays8. The relevance of protein quantitation is evidenced by the presence of two papers describing protein measurement methods5,7 in the top-3 of the most cited literature9,10. Despite the fact that many authors neglect their actual citation by citing non-primary references or not citing anything at all, the original papers describing the Lowry protein assay and the Bradford protein assay amount &#62;200,000 citations each10.
The popularity of the Bradford assay stems from its affordability, simplicity, speed, and sensitivity. The assay is based on the interaction between proteins and the dye Coomassie Brilliant Blue G under acidic conditions. Under the conditions of the assay (i.e., low pH), the dye exists in three forms: a red cationic form with &#955;max at 470 nm; a green neutral form with &#955;max at 650 nm; and a blue anionic form with &#955;max at 590 nm11,12 (Figure 1). The cationic form predominates in the absence of proteins. As proteins interact with the dye, they stabilize the blue anionic form, causing a noticeable change in the color of the solution, from brownish to blue. Usually, the change in the concentration of the blue form of the dye is quantified spectrophotometrically, whose absorbance at 590-595 nm is proportional to the quantity of protein in the assay.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65547/65547fig01v2.jpg" /> 
Figure 1: Coomassie brilliant blue G absorption spectra under the conditions of the Bradford assay. The three main peaks are marked with arrows indicating the &#955;max of the red (470 nm), green (650 nm), and blue (590 nm) forms of the dye. Spectra were recorded in the absence of protein (yellow line) and in the presence of 3 &#181;g (gray line) and 10 &#181;g (blue line) of bovine serum albumin. <a href="https://www.jove.com/files/ftp_upload/65547/65547fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The widespread use of the Bradford assay has led to the identification of several limitations (e.g., variable responses to different proteins11, and interference by lipids13 and detergents7) and the development of modifications to improve its performance (e.g., the addition of detergents14,15, alkalinization14,16 and use of the ratio of absorbances17). In addition to modifications in the assay itself, the use of alternative devices, such as smartphones or cameras, to capture analytical signals have also been described18,19,20. Indeed, the development of methods that make use of smartphones as portable chemical analyzers has been an active area of research. The motivation for the use of smartphones stems from the affordability, portability, ease of use, and widespread availability of these devices.
This paper provides a protocol for protein quantification using the RGBradford assay20, which uses a smartphone as an analytical device. In contrast to the original RGBradford publication20, here, a procedure that streamlines the color extraction process has been introduced. It involves the utilization of a freely available software application to extract color information from each well of a microplate picture automatically, saving significant time and effort. This is an alternative to the previous method of manually acquiring color data from each well one by one using a graphics editor software application20. Ultimately, protein levels in samples can be quantified using color data extracted from a picture of a microplate taken with a smartphone.

作者聚焦：使用智能手机通过 Bradford 分析进行蛋白质定量的另一种方法 

RGBradford：使用智能手机相机进行蛋白质定量

Our research focuses on understanding at the biochemical level how animals adapt to extreme environmental conditions. We explore questions about their response to lack of oxygen and suppression of metabolic rate. Specifically, we investigate endogenous antioxidants, oxidative stress, and cellularity stress response animals employ in a adverse environment.And to do so, we often quantify protein levels in our samples. This protocol addresses the need for an alternative method to obtain protein quantification data when resource are limited, making it a valuable tool in both educational and research contexts. The RG Bradford Assay offers a practical and reliable solution for protein concentration measurement that is accessible, simple, and accurate in scenarios where conventional equipment such as a microplate reader may be unavailable.

Watch this Scientific Journal Video about Automatically Extracting the Bradford Assay Data Recorded with a Smartphone at JoVE.com

Automatically Extracting the Bradford Assay Data Recorded with a Smartphone  

自动提取使用智能手机记录的布拉德福德检测数据

要从使用智能手机相机记录的微孔板图像中提取 Bradford 检测数据，请下载软件图像 J 和以文本文件形式提供的 ReadPlate 插件。打开镜像J后，点击插件，点击安装，选择下载的ReadPlate插件文本文件。通过单击“分析”来设置测量参数，然后单击“设置测量值”并选中以下选项：面积、标准偏差、最小和最大灰度值、平均灰度值、模态灰度值。在窗口底部，将“重定向”设置为“无”，将“小数位”设置为“零”，将“小数位”设置为“9”，设置为“3”。转到文件。单击“打开”，然后选择使用智能手机拍摄的微孔板图片。转到 Plugins，然后转到 ReadPlate。阅读说明，然后单击“确定”。选择孔数为 96。使用插件自动加载的矩形选择工具，从 A1 孔的中心开始，到 H12 孔的中心结束，制作一个矩形，然后单击确定。选择蓝色通道，然后单击确定。然后单击“确定”确认默认参数。检查软件是否在每个孔内划定了一个区域，以及所选区域是否没有覆盖具有异常阴影或反射的区域。选中后，单击“确定”。保存结果，然后对绿色通道重复相同的操作。使用每种颜色的模式计算蓝色与绿色的比率。从微孔板图片自动获得的RGB颜色数据显示，BSA标准品的蓝色值典型增加，红色和绿色值减少。此外，从图片中提取的颜色数据准确地反映了在450和590纳米处记录的吸光度读数。发现使用提取的描绘信号与 BSA 浓度的颜色数据获得的标准曲线是线性的，正如预期的那样。对于吸光度读数以及两种不同蛋白质样品中提取的颜色数据，样品稀释度与信号之间存在线性关系。对于两种蛋白质样品，一些稀释度不在标准曲线的线性范围内。然而，在忽略这些点之后，使用RGB数据计算的两个样品的蛋白质水平与使用吸光度读数计算的蛋白质水平相匹配。

automatically-extracting-bradford-assay-data-recorded-with

Research

JoVE Journal

Biochemistry

Automatically Extracting the Bradford Assay Data Recorded with a Smartphone

267 次观看.  University of Brasilia. 要从使用智能手机相机记录的微孔板图像中提取 Bradford 检测数据，请下载软件图像 J 和以文本文件形式提供的 ReadPlate 插件。打开镜像J后，点击插件，点击安装，选择下载的ReadPlate插件文本文件。通过单击“分析”来设置测量参数，然后单击“设置测量值”并选中以下选项：面积、标准偏差、最小和最大灰度值、平均灰度值、模态灰度值。在窗口底部，将“重定向”设...

视频: Automatically Extracting the Bradford Assay Data Recorded with a Smartphone  

RGBradford: Protein Quantitation with a Smartphone Camera

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. Because of its widespread, the limitations and advantages of the Bradford assay have been exhaustively reported, including several modifications of the original method to improve its performance. One of the alterations of the original method is the use of a smartphone camera as an analytical instrument. Taking advantage of the three forms of the Coomassie Brilliant Blue dye that exist in the conditions of the Bradford assay, this paper describes how to accurately quantify protein in samples using color data extracted from a single picture of a microplate. After performing the assay in a microplate, a picture is taken using a smartphone camera, and RGB color data is extracted from the picture using a free and open-source image analysis software application. Then, the ratio of blue to green intensity (in the RGB scale) of samples with unknown concentrations of protein is used to calculate the protein content based on a standard curve. No significant difference is observed between values calculated using RGB color data and those calculated using conventional absorbance data.

This paper provides a protocol for protein quantification using the Bradford assay and a smartphone as an analytical device. Protein levels in samples can be quantified using color data extracted from a picture of a microplate taken with a smartphone.

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. ...

科研

生物化学

Recording Bradford Protein Assay Results Using a Smartphone Camera

To begin sample preparation for the Bradford protein assay, dilute the protein samples to obtain protein concentrations within the standard curve range of 0.025 to 1.0 milligrams per milliliter. Create multiple sample dilutions per sample within this range. For the zero protein point, add 10 microliters of the buffer or medium used to prepare the standard solutions to the designated wells.Then add 10 microliters of each protein standard solution into a set of three wells serving as triplicates in a 96-well microplate. In a separate set of wells on the same 96-well microplate, add 10 microliters of each sample dilution in triplicates. Next, add 250 microliters of Bradford protein assay reagent to all wells.A layout of the final microplate setup is displayed on the screen. Proceed to record the results in a well illuminated room within 5 to 15 minutes of adding the assay reagent. Using one hand, hold the microplate against a uniform white background, ensuring it is parallel to the work bench.While using the other hand, hold a smartphone parallel to the bench and the microplate. Proceed to capture several pictures of the entire microplate. For iOS devices, enable the grid option in camera settings to turn on the camera level indicator.For Android devices, activate grid lines in camera settings to achieve the same. Although no special lighting apparatus is required, maintain caution while capturing the images properly. Avoid shading the plate or the background and prevent excessive reflection.Also, ensure not to tilt the plate. Inspect the picture for background, uniformity, shadows, and reflections. Also check the angle of the wells:The center of each well should be directly visible If desired, read the microplate in a microplate reader to compare the picture color data with the microplate absorbance readings.

使用智能手机相机记录 Bradford 蛋白检测结果

To extract the Bradford Assay data from the images of the microplate recorded using the smartphone camera, download the software image J and the ReadPlate plugin available as a text file. After opening image J, click on Plugins followed by Install, and select the downloaded ReadPlate plugin text file. Set the measurement parameters by clicking on Analyze, and then Set Measurements and checking the options:Area, Standard deviation, Minimum and maximum gray value, Mean gray value, Modal gray value.On the bottom of the window, set Redirect to as None and Decimal place as zero to 9 as 3. Go to File. Click Open and select the picture of the microplate taken using the smartphone.Go to Plugins followed by ReadPlate. Read the instructions and then click OK.Select the Number of wells as 96. Using the rectangular selection tool automatically loaded by the plugin, make a rectangle beginning in the center of the A1 well and ending in the center of the H12 well, then click OK.Select the Blue channel and click OK.Then confirm the default parameters by clicking on OK.Check if the software delineated an area inside each well, and if the selected areas are not covering areas with abnormal shadows or reflection.Once checked, click OK.Save the results before repeating the same for the Green channel. Calculate the Blue to-Green ratio using the mode for each color. The RGB color data obtained automatically from the picture of a microplate showed the typical increase in the blue values and a decrease in the red and green values for the BSA standard.Further, the color data extracted from the picture accurately reflected absorbance readings recorded at 450 and 590 nanometers. The standard curve obtained with the extracted color data depicting the signal versus BSA concentration was found to be linear as expected. A linear relationship between sample dilution and the signal was obtained for both absorbance readings as well as the extracted color data in two different protein samples.For both protein samples, some of the dilutions were not within the linear range of the standard curve. However, after ignoring those points, protein levels calculated using RGB data for both samples matched those calculated using the absorbance readings.

Manually Extracting the Bradford Assay Data Recorded with a Smartphone

To manually extract the Bradford Assay data from the images of the microplate recorded using the smartphone camera, download Inkscape, a free and open source graphics editor. Open Inkscape, go to file, and click open"to select the picture of the microplate taken using the smartphone. Pick the select and transform objects S tool, which is depicted as an arrow on the top left side, and click on the picture.A dashed line border will indicate the selection. Then select the pick colors from Image D tool, which is depicted as an eyedropper on the left side. Next, click in the center of a well.The color on the bottom panel, fill, will change accordingly. Click on the color and a fill and stroke tab will pop up on the right side. Change the flat color dropdown menu to RGB.Record the values shown for the blue and green channels for each well. Calculate the blue to green ratio using the recorded values.