Name: Automatically Extracting the Bradford Assay Data Recorded with a Smartphone
Uploaded: 2023-09-08T14:50:00
Description: 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

Smartphone as an Analytical Device to Quantify Protein Using Bradford Assay

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.

Author Spotlight: An Alternative Approach to Protein Quantification by Bradford Assay Using a Smartphone 

RGBradford: Protein Quantitation with a Smartphone Camera

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  

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.

Research

JoVE Journal

Biochemistry

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