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.
