recording bradford protein assay results using a smartphone camera

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 Recording Bradford Protein Assay Results Using a Smartphone Camera at JoVE.com

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.

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Research

JoVE Journal

Biochemistry

192 Views.  University of Brasilia. 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 tri...