Laboratory Experiment

pubs.acs.org/jchemeduc

Quantifying Protein Concentrations Using Smartphone Colorimetry:

A New Method for an Established Test
Clifford T. Gee,† Eric Kehoe,‡ William C. K. Pomerantz,*,† and R. Lee Penn*,†
†
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States
‡
Janesville Waldorf Pemberton High School, Janesville, Minnesota 56048, United States
*
S Supporting Information

ABSTRACT: Proteins are involved in nearly every biological

process, which makes them of interest to a range of scientists.
Previous work has shown that hand-held cameras can be used to
determine the concentration of colored analytes in solution, and
this paper extends the approach to reactions involving a color
change in order to quantify protein concentration (e.g., green to
blue). Herein, we describe the successful use of smartphone
colorimetry to quantify protein concentration using two common
colorimetric biochemical methods, the Bradford and biuret assays.
The ease of the experimental setup makes these lab experiments
accessible to a wide range of students and can be used as both
high school and college level laboratory experiments.
KEYWORDS: High School/Introductory Chemistry, First-Year Undergraduate/General, Analytical Chemistry, Biochemistry,
Laboratory Instruction, Hands-On Learning/Manipulatives, Proteins/Peptides, Quantitative Analysis, UV−Vis Spectroscopy

P roteins are large biomacromolecules that play necessary

roles in nearly all biological processes, including cell
growth, metabolism, and differentiation. Consequently, sig-
nificant research efforts are dedicated to studying the biological
function of proteins and designing synthetic molecules to
modulate the function of proteins in the context of disease.
High school students encounter proteins in both the life
sciences as well as in their chemistry courses [e.g., Next
Generation Science Standards (NGSS)1].
One example of an interesting protein is the bromodomain
containing protein 4, (Brd4). When functioning properly, Brd4
is a necessary protein for regulating gene transcription and cell
growth, but when functioning improperly, Brd4 has been linked
to cancer,2 a topic covered in life sciences classes and a reality
that has impacted many people personally.
Accurate quantification of protein concentration is crucial to
studying the roles proteins play in biological processes.
Concentrations are particularly important in biochemical
experiments (e.g., circular dichroism and enzymatic assays)
for quantitative characterization of protein structure and
function. There are several different methods for protein
quantification. Some methods are colorimetric assays that
involve the formation of a colored complex. Quantitation is
accomplished by determining visible light absorption and using
Beer’s Law to convert absorbance to concentration by
comparison to a standard calibration curve. Two such
colorimetric assays are the Bradford and biuret assays.3 Using
known standards within concentration ranges that obey Beer’s
law, one can generate a linear calibration curve that can be used
to estimate a protein concentration of an unknown sample.
© 2017 American Chemical Society and
Division of Chemical Education, Inc.
These methods are especially useful when the protein
concentration in solution cannot be determined by other
techniques e.g., when the protein extinction coefficient is
unknown or is too low for direct determination of the
concentration based on the protein solution’s absorbance at
280 nm.4
Colorimetric assays are typically performed using a UV−vis
spectrophotometer. In the presence of varying concentrations
of protein, absorbance values at 595 and 540 nm are monitored
in the Bradford and biuret assays, respectively. However, these
specialized instruments are expensive and may not be available
in all laboratories such as high school teaching laboratories. To
mitigate this challenge, recent reports have demonstrated the
efficacy of hand-held cameras for quantitative colorimetry,
making colorimetric experiments accessible to a wider range of
students and researchers.5 Smartphone colorimetry has been
used in kinetics experiments with crystal violet,6 quantification
of gold nanoparticles present in dietary supplements,7 and a
variety of other experiments.8−12 These examples involve a
single color with a colorless blank. The use of camera phone
colorimetry with a color change has yet to be explored. Here,
we extend quantitative colorimetry to protein assays that
involve color changes and its application to quantify protein
concentration.
Bradford assays are commonly employed in laboratory
experiments for introductory biochemistry courses. The
Received: September 6, 2016
Revised: April 6, 2017
Published: May 3, 2017
941 DOI: 10.1021/acs.jchemed.6b00676
J. Chem. Educ. 2017, 94, 941−945
Journal of Chemical Education
Figure 1. Chemical scheme and sample images for protein colorimetric assays. (A) Chemical scheme for Coomassie Blue G-250 which becomes fully
deprotonated in the presence of a protein, yielding a blue color. (B) Sample image of Bradford reagent plus water (left) and Bradford reagent plus
0.04 mg/mL bovine serum albumin (BSA) (right). (C) Chemical scheme for complexation of cupric ions in the presence of peptides and proteins
forming a complex with the amide backbone, resulting in a color change from blue to violet. (D) Sample image of biuret reagent plus water (left) and
biuret reagent plus 0.75 mg/mL BSA (right).
protein-responsive molecule in the Bradford solution, Coo-
massie Blue G-250, is an environmentally sensitive triphenyl-
methane dye compound that exists in varying charge states
depending on the surrounding solution conditions. The
hydrophobic interactions provided by the presence of a protein
stabilizes the anionic form of the dye, resulting in color change
from a brownish green, with a maximum absorption at 470 nm
(cationic form), to a blue color, with a maximum absorption at
595 nm (anionic form) (Figure 1A,B).
While the Bradford assay is a dye-based assay, the biuret
assay involves the formation of a Cu(II) complex with the
amide backbone present in peptides and proteins. This
coordination complex results in a color change from blue
(free Cu2+), with a maximum absorption at 650 nm, to violet
(HN···Cu2+···NH), with a maximum absorption at 540 nm
(Figure 1C,D). The bicinchoninic acid (BCA) and Lowry
assays are examples of other copper-based assays that are
commercially available.
Herein we report the first application of smartphone
colorimetry for protein quantification using two well-known
protein assays that involve changes in color as a function of
protein concentration. The ubiquity of the Bradford assay as a
standard laboratory experiment in biochemistry and the cheap
and accessible nature of the biuret assay combined with the
clear color change that occurs upon complexing with protein
made these two assays ideal for use with smartphone
colorimetry.
■ MATERIALS AND METHODS
Preparation of Chemicals
The Bradford reagent was prepared with Coomassie Blue G-
250 (Sigma), methanol (Sigma), phosphoric acid (Macron),
and water according to the following protocol: (1) Dissolve 50
mg Coomassie Blue G-250 in 50 mL methanol. (2) Add 100
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Laboratory Experiment
mL 85% (w/v) phosphoric acid. (3) Add this mixture slowly to
850 mL of water. (4) Store away from light at 4 °C. Because
Flinn is a common vendor for high school laboratories, the
biuret reagent was prepared using the Flinn Scientific
specifications [0.2% (w/v) CuSO4, 30% (w/v) NaOH] for
this reagent. This solution can be made by mixing 100 mg of
CuSO4·5H2O (dissolved in 10 mL of water) and 40 mL of
37.5% (w/v) NaOH in water. It is important to note that biuret
from Flinn contains 30% sodium hydroxide while many other
vendors and recipes employ only 3% (w/v) sodium hydroxide
with the addition of 0.6% (w/v) potassium sodium tartrate.13
Both solutions will provide adequate results, but the tartrate-
containing solution performs significantly better than the
solution without it at protein concentrations above 0.5 mg/
mL because it prevents protein precipitation. If tartrate is not
available, citrate may also be used as a substitute to prevent
turbidity (see Supporting Information). While the Bradford
reagent is more common in biochemistry laboratories for its
greater sensitivity to a wider protein concentration range, biuret
is roughly 10% of the cost of Bradford, making it more
accessible for a high school or a low-budget laboratory. Bovine
serum albumin (BSA) for the calibration curve was purchased
from RPI and used as-is. Brd4 was expressed from recombinant
DNA using standard protein expression protocols in BL21
(DE3) E. coli cells.14
Preparation of Calibration and “Unknown” Solutions
Standard Bradford or biuret solutions for the calibration curve
were prepared by diluting 0.10 mg/mL BSA (for Bradford
assay) or 3−10 mg/mL BSA (for biuret assay) with 1.5 mL of
Bradford or biuret solution and water. For preparation of the
calibration standards, 1.5 mL of the Bradford or biuret
solutions; 0.1, 0.2, 0.3, 0.4, and 0.5 mL of appropriate protein
stock solution; and enough water to yield a final volume of 2.0
mL were added to individual vials. The blank sample was
prepared by mixing 1.5 mL of the Bradford or biuret and 0.5
DOI: 10.1021/acs.jchemed.6b00676
J. Chem. Educ. 2017, 94, 941−945
Journal of Chemical Education Laboratory Experiment

Figure 2. Photographs of calibration samples plus an unknown sample for smartphone colorimetry. (A) Biuret standards for calibration and
“unknown” sample in front of a white background. (B) Biuret standards for calibration and “unknown” sample in front of a green (129, 255, 0)
background. (C) Biuret standards for calibration and “unknown” sample in front of a yellow-orange (255, 207, 0) background.

Figure 3. Comparison of colorimetric assays by UV−vis spectrophotometry and smartphone colorimetry. (A) Photograph of Bradford assay cuvettes
in front of 595 nm (RGB: 255, 207, 0) colored background. (B) Graph of absorbance (via spectrophotometry) versus protein concentration. (C)
Graph of absorbance (via smartphone colorimetry following R values) versus protein concentration. (D) Photograph of biuret assay cuvettes in front
of 540 nm (RGB: 129,255,0) colored background. (E) Graph of absorbance (via spectrophotometry) versus protein concentration. (F) Graph of
absorbance (via smartphone colorimetry following G values) versus protein concentration. In all plots, blue diamonds refer to protein calibration,
and the red circle indicates the “unknown” sample at its expected concentration.

mL of water. Experiments can all be performed with regular tap
water. As noted in the sample student guide (Supporting
Information), these low volumes of protein solution can be
delivered into the individual square plastic cuvettes (1 cm path
length) by counting drops of the solution and weighing the
sample using a balance after each addition to account for the
actual amounts added. This approach works well since the
densities of each solution and water are all nearly identical.
Further, if students do not have access to a balance, counting
drops and converting volume to mass can also provide adequate
data, as noted in the Results section. Absorbance measurements
were performed 40 min after mixing for the Bradford and 20
min after mixing for the biuret samples in order to ensure full
development of the color change. The “unknown” protein
concentration sample was prepared in a similar manner, with
the estimated concentration falling within the linear range of
the colorimetric agents. The “unknown” protein concentration
in the sample Bradford assays was 0.014 mg/mL Brd4 as
determined by UV absorption (ε280: 26,930 M−1 cm−1) and
1.25 mg/mL BSA by mass in the sample biuret assay. Because
the change in color is a time-dependent process, the
943
equilibration time is important for the color to fully develop.
Insufficient equilibration time will often lead to poorer results.
An example of this time-dependent effect is shown in the
teacher’s guide in the Supporting Information.
Instrumentation and Setup
Cuvettes containing the standard, blank, and “unknown”
samples were analyzed using both a standard UV−vis
spectrophotometer and smartphone colorimetry. For absorp-
tion measurements, samples were analyzed at 595 nm for
Bradford and 540 nm for biuret using a Beckman Coulter DU
720 UV−vis spectrophotometer.
Acquiring Images
For smartphone colorimetry, cuvettes were lined up in front of
a computer monitor, and images were acquired using the back
facing lens of varying smartphones (Samsung Galaxy S5,
iPhone 6). Cuvettes were lined up in front of a computer
monitor with varying background colors matching the wave-
lengths measured for absorption in addition to a white
background (Figure 2). For Bradford samples, an RGB code
of (255, 207, 0) was used, while (129, 255, 0) was used for the
DOI: 10.1021/acs.jchemed.6b00676
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Journal of Chemical Education
biuret. These values can be obtained using an online
wavelength to RGB conversion tool (e.g., Academo’s Wave-
length to Color Relationship15).
Data Analysis and Comparison
Absorption data from the spectrophotometer were plotted
using spreadsheet software and fitted to a linear trendline. The
generated equation was used to estimate the protein
concentration. Absorbance measurements from the smartphone
images were obtained by analyzing RGB (red, green, blue)
values from images of each sample (intensity range 0−255). A
variety of software, freeware, and web applications (e.g., Adobe
Photoshop, ImageJ, Color Code Picker)5 as well as smartphone
applications (e.g., Colorometer for iPhone and ColorMeter
Free for Android)11 are available for this type of analysis and
provide similar results. Download links can be found in the
Supporting Information. RGB values can be obtained by
analyzing single points or averaging multiple points over a
specified area. Single point and area measurements yield nearly
identical results, as noted in a previous report.5
Absorbance (A) is calculated from RGB values using the
following formula:
⎛ I ⎞
A = −log⎜ n ⎟
⎝ Iblank ⎠ (1)
Here, In corresponds to the R, G, or B value of each sample,
and Iblank corresponds to the R, G, or B value for the blank. For
the Bradford assay, the best results were obtained using the R
values while for the biuret assay, the best results were obtained
using the G values. For alternative colorimetric experiments or
assays, it would be best to follow whichever channel (R, G, or
B) provides the greatest dynamic range. These “absorbance”
values were plotted as a function of concentration and fit to a
linear trendline to estimate protein concentration of the
“unknown” sample.
■ HAZARDS
The Bradford solution is acidic while the biuret solution is
basic. Both are corrosive and should be handled with care. Both
solutions should be neutralized appropriately prior to disposal
down the drain with excess water. Alternatively, solutions can
be disposed of in separate waste containers as appropriate.
Protective gloves and eyewear (preferably splash proof goggles)
in addition to standard lab-appropriate attire (long pants and
closed-toed shoes) should be worn at all times while handling
Bradford or biuret reagents.
■
RESULTS
Samples from both the Bradford and biuret assays were
evaluated by a UV−vis spectrophotometer and by smartphone
colorimetry to benchmark a newer technique against an
established technique. Calibration curves generated R2 values
greater than 0.9, and using the equation generated from fitting a
linear trendline, the concentration of protein in the “unknown”
sample was calculated (Figure 3).
■
DISCUSSION
The use of a smartphone camera is effective for quantitative
colorimetry measurements. It can be used for the appearance of
color, as previously reported,5−10 and also for a color change, as
described here. The obtained data were adequate for correctly
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estimating a given protein concentration using Beer’s Law and a
standard calibration curve.
Smartphone colorimetry data were comparable to data
collected using the spectrophotometer (Figure 3B,C). In the
example of the Bradford assay above (Figure 3A−C), the
“unknown” concentration was 0.014 mg/mL, and the UV−vis
absorption measurements yielded an estimated concentration
of 0.011 mg/mL while a concentration of 0.0090 mg/mL was
estimated via camera phone colorimetry. In the example of the
biuret assay (using the tartrate-containing solution) (Figure
3D−F), the “unknown” concentration was 1.25 mg/mL, and
the UV−vis absorption measurements yielded an estimated
concentration of 1.32 mg/mL while a concentration of 1.28
mg/mL was estimated via camera phone colorimetry. Images
acquired in front of backgrounds displaying the color matching
the maximum absorption wavelength of the color agent yielded
more accurate results as compared to images of the samples
arranged in front of a white display. However, if a backlit
background is not available, a plain white background is suitable
for collecting quantitative measurements. Kuntzleman and
Jacobson also reported good success using colored construction
paper in lieu of a colored backlit screen for similar types of
measurements.11
The utility of this method lies not only in its efficacy for
determining protein concentration via the measurement of a
color change but also in its robustness to a range of factors,
including lighting conditions and picture quality, which can
vary dramatically from one classroom to another. Given the
camera quality found in the average smartphone, this type of
protein quantification colorimetry experiment is now more
widely accessible to students who may have previously been
unable to perform such an experiment.
As a final assessment of the suitability of the experiment for a
high school classroom as well as the clarity of the instructions
for the experiment, the method was tested with a group of
tenth grade high school chemistry students. The lab was
conducted with two different class sizes of 25 and 15 students,
respectively, and run over three 50 min class periods. The first
class period was used to introduce the background for the lab
and demonstrate the experimental procedure which can be
found in the Supporting Information. The second period was
used to prepare the solutions and capture the image using
smartphones. During the last day, the images were analyzed and
the results discussed.
In order to ensure the method is easily reproducible in any
high school setting, a few changes were made from the original
protocol. For high school implementation we used the biuret
and albumin as they are available from Flinn Scientific. Also,
because many laboratories may not have access to protein
expression facilities, using an unknown concentration of
albumin, or other commercially available proteins (e.g.,
lysozyme or cytochrome C), is a more practical alternative
than Brd4. For this study, six groups had access to electronic
balances with centigram precision. In order to balance time
efficiency and reagents, the students worked in groups of five.
The two groups without access to balances were provided with
a conversion from drops of solution to mass for each of the
solutions.
Analysis of the students’ data pointed to several areas for
further clarification in the protocol. At this point, all students
had demonstrated a strong grasp of Beer’s law; however,
analysis of the results show that only four of the eight groups
produced an unknown concentration that agreed within 10% of
DOI: 10.1021/acs.jchemed.6b00676
J. Chem. Educ. 2017, 94, 941−945
Journal of Chemical Education
the unknown protein concentration. The remaining four groups
had considerably larger errors. It should be noted that positive
results were independent of sample preparation method. In the
four groups that yielded accurate results and in the four that
yielded poor data, three groups prepared samples with a
balance and one group counted drops. After reviewing the
students’ work, it became clear that some groups whose analysis
fell outside of 10% of the accepted concentration had not taken
images of suitable quality for analysis. A common error was
taking the picture from too far away, leaving the color intensity
in the image washed out, as the area of interest was very bright
relative to the background. Poor image focus also led to
unreliable data. To alleviate these problems for future
implementation, examples of good and poor images for
students have now been added to the instructional guide
which can be found in the Supporting Information.
In general, the instructor felt the results were of similar
quality to those from other quantitative methods carried out by
high school students at this level. The use of photocolorimetry,
Beer’s Law, and protein topics aligns well with courses
introducing content that uses the Next Generation Science
Standards, HS-LS1-1, regarding content on protein function, as
well as HS-PS4-5 which describes how some technological
devices use the principles of wave behavior and wave
interactions with matter to transmit and capture information
and energy.
■ CONCLUSION
Smartphones and other portable cameras were successfully used
to determine the concentration of a protein sample using assays
that yield a color change as a function of protein concentration.
This new method for measuring protein concentrations allows
for greater accessibility to such colorimetric assays for
introductory laboratories and laboratories lacking expensive
equipment like spectrophotometers. This type of experiment
also fits in well with the Science and Engineering Practices from
the HS-LS1 section of the NGSS.1 A detailed step-by-step
procedure for the experiment suitable for use in high school or
college settings is provided in the Supporting Information.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available on the ACS
Publications website at DOI: 10.1021/acs.jchemed.6b00676.
Sample student guide, providing step-by-step instruc-
tions, as well as some instructor notes (PDF, DOCX)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail: wcp@umn.edu.
*E-mail: rleepenn@umn.edu.
ORCID
William C. K. Pomerantz: 0000-0002-0163-4078
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
The authors would like to thank Alex Ayoub for his suggestion
of substituting potassium sodium tartrate with sodium citrate as
a cheaper alternative reagent. This project was funded in part
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by the NSF-CAREER Award CHE-1352091. C.T.G. would like
to acknowledge the University of Minnesota Interdisciplinary
Doctoral Fellowship.
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