Name: Author Spotlight: An Alternative Approach to Protein Quantification by Bradford Assay Using a Smartphone
Uploaded: 2023-09-08T14:50:00
Description: Watch this Scientific Journal Video about An Alternative Approach to Protein Quantification by Bradford Assay Using a Smartphone at JoVE.com

This work was funded by the National Council for Scientific and Technological Development (CNPq, Brazil) [grant numbers 428048/2018-8 and 402556/2022-4] and the University of Brasilia (Brazil).&#160;The author thanks Dr. Duarte Nuno Carvalho and Dr. Evelyn Santos (i3s, Porto, Portugal) for providing access to their smartphones used in this research.

1. Preparation of the Bradford protein assay reagent
<ol>
	<li>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.</li>
	<li>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. ...

Smartphone as an Analytical Device to Quantify Protein Using Bradford Assay

<ol>
	<li>Zaguri, M., Kandel, S., Rinehart, S. A., Torsekar, V. R., Hawlena, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+quantification+in+ecological+studies:+A+literature+review+and+empirical+comparisons+of+standard+methodologies.">Protein quantification in ecological studies: A literature review and empirical comparisons of standard methodologies.</a> Methods in Ecology and Evolution. 12 (7), 1240-1251 (2021).</li><li>Koga, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild+electrical+stimulation+and+heat+shock+ameliorates+progressive+proteinuria+and+renal+inflammation+in+mouse+model+of+Alport+syndrome.">Mild electrical stimulation and heat shock ameliorates progressive proteinuria and renal inflammation in mouse model of Alport syndrome.</a> PLoS One. 7 (8), e43852 (2012).</li><li>Peterson, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+total+protein.">Determination of total protein.</a> Methods in Enzymology. 91, 95-119 (1983).</li><li>Goldfarb, A. R., Saidel, L. J., Mosovich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ultraviolet+absorption+spectra+of+proteins.">The ultraviolet absorption spectra of proteins.</a> The Journal of Biological Chemistry. 193 (1), 397-404 (1951).</li><li>Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+measurement+with+the+Folin+phenol+reagent.">Protein measurement with the Folin phenol reagent.</a> The Journal of Biological Chemistry. 193 (1), 265-275 (1951).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Analytical Biochemistry. 150 (1), 76-85 (1985).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72 (1-2), 248-254 (1976).</li><li>Datki, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+BisANS+fluorescent+dye+for+developing+a+novel+protein+assay.">Application of BisANS fluorescent dye for developing a novel protein assay.</a> PLoS One. 14 (4), e0215863 (2019).</li><li>Van Noorden, R., Maher, B., Nuzzo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+top+100+papers.">The top 100 papers.</a> Nature. 514 (7524), 550-553 (2014).</li><li>. Scopus Available from: <a target="_blank" href="https://www.scopus.com/">https://www.scopus.com/</a> (2022)</li><li>Compton, S. J., Jones, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+dye+response+and+interference+in+the+Bradford+protein+assay.">Mechanism of dye response and interference in the Bradford protein assay.</a> Analytical Biochemistry. 151 (2), 369-374 (1985).</li><li>Chial, H. J., Thompson, H. B., Splittgerber, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spectral+study+of+the+charge+forms+of+Coomassie+Blue+G.">A spectral study of the charge forms of Coomassie Blue G.</a> Analytical Biochemistry. 209 (2), 258-266 (1993).</li><li>Pande, S. V., Murthy, M. S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+micro-Bradford+procedure+for+elimination+of+interference+from+sodium+dodecyl+sulfate,+other+detergents,+and+lipids.">A modified micro-Bradford procedure for elimination of interference from sodium dodecyl sulfate, other detergents, and lipids.</a> Analytical Biochemistry. 220 (2), 424-426 (1994).</li><li>Gogstad, G. O., Krutnes, M. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+in+cell+suspensions+using+the+Commassie+brilliant+blue+dye-binding+assay.">Measurement of protein in cell suspensions using the Commassie brilliant blue dye-binding assay.</a> Analytical Biochemistry. 126 (2), 355-359 (1982).</li><li>Friedenauer, S., Berlet, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+and+variability+of+the+Bradford+protein+assay+in+the+presence+of+detergents.">Sensitivity and variability of the Bradford protein assay in the presence of detergents.</a> Analytical Biochemistry. 178 (2), 263-268 (1989).</li><li>Stoscheck, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+uniformity+in+the+response+of+the+Coomassie+blue+G+protein+assay+to+different+proteins.">Increased uniformity in the response of the Coomassie blue G protein assay to different proteins.</a> Analytical Biochemistry. 184 (1), 111-116 (1990).</li><li>Zor, T., Selinger, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linearization+of+the+Bradford+protein+assay+increases+its+sensitivity:+Theoretical+and+experimental+studies.">Linearization of the Bradford protein assay increases its sensitivity: Theoretical and experimental studies.</a> Analytical Biochemistry. 236 (2), 302-308 (1996).</li><li>Gee, C. T., Kehoe, E., Pomerantz, W. C. K., Penn, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+protein+concentrations+using+smartphone+colorimetry:+A+new+method+for+an+established+test.">Quantifying protein concentrations using smartphone colorimetry: A new method for an established test.</a> Journal of Chemical Education. 94 (7), 941-945 (2017).</li><li>de Camargo, C., Vicentini, M., Gobbi, A., Martinez, D., Lima, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smartphone+for+point-of-care+quantification+of+protein+by+Bradford+assay.">Smartphone for point-of-care quantification of protein by Bradford assay.</a> Journal of the Brazilian Chemical Society. 28 (4), 689-693 (2016).</li><li>Moreira, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGBradford:+Accurate+measurement+of+protein+concentration+using+a+smartphone+camera+and+the+blue+to+green+intensity+ratio.">RGBradford: Accurate measurement of protein concentration using a smartphone camera and the blue to green intensity ratio.</a> Analytical Biochemistry. 655, 114839 (2022).</li><li>Ernst, O., Zor, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linearization+of+the+Bradford+Protein+Assay.">Linearization of the Bradford Protein Assay.</a> Journal of Visualized Experiments. (38), 1918 (2010).</li><li>Angelani, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+metabolic+control+analysis+approach+to+introduce+the+study+of+systems+in+biochemistry:+the+glycolytic+pathway+in+the+red+blood+cell:+Metabolic+control+analysis+and+the+glycolytic+pathway.">A metabolic control analysis approach to introduce the study of systems in biochemistry: the glycolytic pathway in the red blood cell: Metabolic control analysis and the glycolytic pathway.</a> Biochemistry and Molecular Biology Education. 46 (5), 502-515 (2018).</li></ol>

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, ...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well flat-bottom polystyrene microtiter plates&#160;</td>
			<td>Jet Biofil, Guangzhou, China</td>
			<td>TCP011096</td>
			<td>Any flat-bottom microplate compativle with optical reading will suffice.&#160;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue G</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>B0770</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iPhone 11</td>
			<td>Apple</td>
			<td>MWM02BR/A</td>
			<td>Can be substituted with other smartphone equiped with a camera</td>
		</tr>
		<tr>
			<td>iPhone 14 Pro</td>
			<td>Apple</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric acid</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>695017</td>
			<td></td>
		</tr>
		<tr>
			<td>Redmi Note 9 Pro</td>
			<td>XIAOMI&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>S22 Ultra</td>
			<td>Samsung&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax 384 Plus. Microplate reader.</td>
			<td>Molecular Devices, San Jose, CA</td>
			<td>PLUS 384</td>
			<td>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.</td>
		</tr>
	</tbody>
</table>

rgbradford: protein quantitation with a smartphone camera

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.

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.

Figure 4&#160;is a picture of a microplate from which color data was extracted, and absorbance at 450 nm and 590 nm was recorded. The RGB color data reported here as representative were obtained automatically as described in section 5. A typical pattern of color data is an increase in the blue values and a decrease in the red and green values (Figure 5). Note that despite the evident reflection in all wells and a not perfectly aligned microplate (<strong class="...

Watch this Scientific Journal Video about An Alternative Approach to Protein Quantification by Bradford Assay Using a Smartphone at JoVE.com

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.

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