RGBradford: Protein Quantitation with a Smartphone Camera

Summary

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.

Abstract

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.

Introduction

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 readings

Protocol

1. Preparation of the Bradford protein assay reagent

1. Dissolve 100 mg of Coomassie Brilliant Blue G in 50 mL 95% (w/v) ethanol. Mix until Coomassie Brilliant Blue G is completely dissolved.
   CAUTION: Ethanol is flammable and causes eye irritation. Avoid flames and use goggles.
2. To the previous solution, add 100 mL of 85% (w/v) phosphoric acid carefully.
   CAUTION: Phosphoric acid is corrosive to metals and causes skin corrosion, serious eye damage, and acute oral toxicity. .......

Discussion

This paper describes RGBradford, a method that uses a smartphone camera to record data from a Bradford protein assay, extract color data, and accurately quantify protein levels in biological samples as originally described recently²⁰. One difference from the original RGBradford method is that here a procedure for obtaining color data automatically with an ImageJ plugin²² was used. The main novelty of the RGBradford method is the use of RGB data as analytical signals; thus, .......

Materials

Name	Company	Catalog Number	Comments
96-well flat-bottom polystyrene microtiter plates	Jet Biofil, Guangzhou, China	TCP011096	Any flat-bottom microplate compativle with optical reading will suffice.
Bovine serum albumin	Sigma-Aldrich, St. Louis, MO	A2153
Coomassie Brilliant Blue G	Sigma-Aldrich, St. Louis, MO	B0770
Ethyl alcohol
iPhone 11	Apple	MWM02BR/A	Can be substituted with other smartphone equiped with a camera
iPhone 14 Pro	Apple	N/A
Phosphoric acid	Sigma-Aldrich, St. Louis, MO	695017
Redmi Note 9 Pro	XIAOMI	N/A
S22 Ultra	Samsung	N/A
SpectraMax 384 Plus. Microplate reader.	Molecular Devices, San Jose, CA	PLUS 384	Any microplate reader capable of reading at 450 nm and 590 nm will work. This is optional. The method was actually created to dismiss the need of a microplate reader.