Our research focuses on understanding at the biochemical level how animals adapt to extreme environment and conditions. We explore questions about their response to lack of oxygen and suppression of metabolic rate. Specifically, we investigate endogenous antioxidants, oxidative stress, and cellular stress response animals employ in 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 available in both educational and research contexts. The RG breadth for 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. 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 five 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. To extract the Bradford assay data from the images of the microplate recorded using the smartphone camera, download the software ImageJ and the read plate plugin available as a text file.
after opening ImageJ, click on plugins followed by install, and select the downloaded read plate plugin text file. Set the measurement parameters by clicking on analyze, and then set measurements in 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 places zero to nine as three.
Go to file, click open, and select the picture of the microplate taken using the smartphone. Go to plugins, followed by read plate. Read the instructions and then click okay.
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 okay. Select the blue channel and click okay then confirm the default parameters by clicking on okay.
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 okay. 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 dilution 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. 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.
