Group 5 Date Submitted: September 26, 2018

de Leon, Lavarro, Leyble, Pineda, Resurreccion Date Performed: September 19, 2018

Exercise 3
Colorimetric Analysis of Protein Concentration (Bradford Assay)

Abstract
The experiment focused on the concept of colorimetric analysis of protein
concentration using the Bradford assay method. The objective of the experiment is for the
students to be able to perform the Bradford assay method for colorimetric determination of
the unknown protein concentration of the unknown. Likewise, students must be able to
describe the principle of ultraviolet absorption and how it is applied to determine the
concentration of protein, as well as to explain the significance of determining the
concentration. The experiment was conducted by first setting the wavelength of the
spectrophotometer to 595 nm. The blank was then prepared, and the Bradford Reagent was
used. A 1 cm path length cuvette was used to measure the absorbance at 595 nm (Abs​595​).
The absorbance values versus the protein concentration were plotted to make the standard
curve. Lastly, the unknown was prepared along with the said reagent. The Abs​595 of the
unknown sample protein concentration was measured. The concentration was computed
using the standard curve prepared. The group’s results show that the amount of protein in
the unknown solution is 36.26 μg. There may be a deviation from the accepted value of the
concentration of the unknown, which is 35 μg, due to human and instrumental error. This
resulted to a percent error of 3.6%. Despite this, however, the group was still able to fulfill its
objectives of applying the Bradford assay along with the prepared standard curve to identify
the protein concentration of the unknown, as well as knowing the significance of determining
the protein concentration.

I. Introduction
Colorimetry simply refers to the measurement of color, which is defined as “the
quality of an object or substance with respect to light reflected by the object, usually
determined visually by measurement of hue, saturation, and brightness of the reflected light”
(Khan Academy 2016). In line with the aforementioned, colorimetric analysis is a method
used in determining the concentration of a chemical element or compound in a solution
based on relative absorption of light with the aid of a color reagent (Constable and
Housecroft 2006). This can be done given that numerous biological molecules inherently
absorb light in the ultraviolet spectrum. Two fundamental laws are primarily applied in this
process: Lambert’s Law, which relates the amount of light absorbed and the distance it
travels through an absorbing medium; and Beer’s Law, which relates light absorption and the
concentration of the absorbing substance (The Editors of Encyclopedia Britannica 2014).
These two laws may be combined to form the Beer-Lambert Law, which describes the
quantitative relationship in the absorption process, as seen in the equation below:

Equation 1. Beer-Lambert Law

I = I​o10
​
-εcd
​

Where ​lo​ = intensity of the incident radiation; ​I = intensity of the radiation transmitted
through a cell of thickness ​d (in centimeters) that contains a solution of concentration ​c (in M
or grams/100mL) and ε is an absorptivity constant (extinction coefficient) characteristic to the
substance being investigated.
In addition to this, the absorbance ​Abs is directly related to ​I​o ​and ​I​, wherein it is
directly proportional to concentration as seen in the equation below:
 Equation 2. Absorbance Equation
​ (​ IoI )
Abs = log10
Abs = εcd

The Bradford Assay, which is a colorimetric analysis, is a method that involves the
binding of Coomassie Brilliant Blue G-250 to the protein being analyzed. The binding of the
Coomassie Brilliant Blue G-250 to the protein causes a shift in the absorption maximum of
the dye from 465 nm to 595 nm, wherein it is the increase in absorption at 595 nm that is
being monitored (Bradford 1976). This assay is recommended for general use especially for
determining the protein content of cell fractions and assessing protein concentrations. In
addition to the aforementioned, this assay is useful since the extinction coefficient, ​ε,​ of a
dye-albumin complex solution remains constant over a 10-fold concentration range.
Furthermore, it is easier to observe because of the visible color change that occurs due to
the hydrophobic and ionic interactions that stabilize the anionic form of the dye; it is also
easier to reproduce given that the dye binding process is virtually complete in approximately
two minutes with good color stability for one hour (Bradford 1976).
In this experiment, the concentration of an unknown protein will be determined with
the use of absorption-visible wavelengths based on the protein binding to a dye. In order to
do this, a standard curve is first prepared by measuring the absorbance of a protein standard
of known concentration, usually that of bovine serum albumin or BSA. This standard is a
globular protein and is often used in numerous biochemical applications due to its stability
and lack of interference within biological reactions (Sigma-Aldrich). With that, the
concentration of the unknown sample can then be determined graphically from the standard
curve.
The objectives of the experiment are to perform the Bradford assay in order to
determine the concentration of the unknown protein using the prepared standard curve; to
describe the principle and application of ultraviolet absorption in relation to the
aforementioned; and to explain the significance of determining the protein concentration of a
substance. Overall, the aim of the experiment is to properly perform the assay and
determine the unknown substance while applying the concepts of colorimetry and
colorimetric analysis.
The process of determining the protein concentration of a substance plays a
significant role in many different fields of science and medicine. For example, analyzing the
interactions between biomolecules is essential for understanding their function; however,
prior to this, the concentration must first be determined in order to have an accurate
measurement of the kinetic interaction between the two interacting proteins. In addition to
this, the characterization of binding proteins to other proteins, to nucleic acids, or to other
small molecules is fundamental to biochemical research as well as other areas, including
that of drug discovery and biopharmaceuticals (Pol 2010).

II. Methodology
The first part of the experiment deals with preparing the standard curve. Before
starting, the spectrophotometer was turned on and set to 595 m. In order to prepare the
standard curve, five aliquots of 1.0 mg/mL BSA were prepared in 1.5 mL microcentrifuge
tubes with the following protein concentrations: 5.0 μL, 10.0 μL, 25.0 μL, 50.0 μL and 75.0
μL. Using 0.15 M NaCl, the volume of each test tube was brought to a total of 100 μL. In
order to calibrate the spectrophotometer, a blank or a solution containing little to no analyte
was prepared by adding 100 μL of 0.15 M NaCl into a 1.5 mL microcentrifuge tube. 1 mL of
Bradford Reagent was added to each test tube. They were then vortexed and left to rest for
two minutes at room temperature. Before reading the five aliquots of 1.0 mg/mL BSA, the
absorbance at 595 nm (Abs​595​) of the blank was recorded in order to standardize the
spectrophotometer. Once the blank’s Abs​595 was read, the Abs​595 of each of the test tubes
were measured using a 1 cm path length cuvette starting from the most dilute until the most
concentrated. It is important that the cuvette was rinsed with ethanol and ddH​2​O before each
reading. The Abs​595 of each aliquot was equal to the reading given by the spectrophotometer
subtracted by the Abs​595 of the blank. To create the standard curve, the results were inputted
into Microsoft® Excel, and a standard curve in the form of a line graph was plotted.
Once the absorbance versus total proteins (μg) graph of the standard curve was
created, the unknown was prepared. In a 1.5 mL microcentrifuge tube, 1 mL of Bradford
reagent was added to 100 μL of the unknown protein. The solution was then vortexed and
left to rest for two minutes at room temperature. The Abs​595 of the unknown was measured
by the spectrophotometer. The Abs​595 of the blank was then subtracted from the reading
given by the spectrophotometer in order to arrive at the Abs​595 of the unknown. Using the
equation of the line given by the graph created in Microsoft® Excel, the protein concentration
of the unknown was determined.

III. Results & Discussion

Figure 1. Bradford Assay Concentration vs Absorbance Standard Curve

*The 75.0 reading was removed from the standard curve for the trendline to better fit the other data.

Table 1. Spectrophotometer Readings and Abs​595​ of Unknown

Spectrophotometer Reading Abs​595​ of Blank Abs​595​ of Unknown

1.681 0.919 0.762

Table 2. Tabulation of Standard Curve Data and Unknown

Tube Vol. of 1.0 mg/mL μg BSA Abs​595
BSA added
 Blank 0.0 0.0 0.919

1 5.0 5.0 0.179

2 10.0 10.0 0.304

3 25.0 25.0 0.549

4 50.0 50.0 1.012

5 75.0 75.0 1.195

6 Unknown μL 36.26 0.762

*The absorbance value of the blank has already been subtracted from the Abs​595​ spectrophotometer readings on Tubes 1-6.

In this experiment, the Bradford assay method was used in determining the
concentration of an unknown protein. According to Kruger’s “The Bradford Method for
Protein Quantitation” article in ​The Protein Protocols Handbook,​ the Bradford assay relies on
the binding of the Coomassie Brilliant Blue G-250 to the protein, such that the levels of the
dye detected are proportional to the amount of protein present. Comprehensive studies
show that the aforementioned dye can exist in four different ionic forms, wherein of the three
charged forms that predominate the acidic solution, the more cationic red and green forms
have an absorbance maxima of 470 nm and 650 nm, respectively. In contrast to this, the
more anionic blue form of the dye, which essentially binds to the protein, has an absorbance
maximum of 590 nm. Thus, the protein concentration can be estimated by determining the
amount of dye found in the blue ionic form; this is usually obtained by measuring the
absorbance of the solution at 595 nm (Walker 2002) (Answer to Q2).
The results in Table 2 as well as Figure 1 demonstrate the quantitative relationship in
the absorption processes; that is, there is a linear relationship between absorbance and
concentration. To determine the amount of protein in the solution, the group used a UV-VIS
spectrophotometer to provide the absorbance value of the solution at 595 nm, which was
1.681, as seen in Table 1. The absorbance of the blank was then subtracted from this value
in order to obtain the absorbance of the unknown, which is 0.762. With that, the amount of
protein in the solution was computed using the trendline equation of the standard curve. The
amount of protein in the unknown solution was found to be 36.26 μg (Answer to Q1). Given
that the accepted value is 35 μg, the obtained experimental value was accurate, with only a
3.6% error.
It is important to note that the group removed the data for Tube 5 in the linear
regression because it was an outlier compared to the rest of the data; adding the Tube 5 μg
BSA and Abs​595 data to the standard curve would distort the resulting trendline and give an
inaccurate result in computing for the amount of protein in the unknown solution.
The results from this particular experiment should be interpreted within the context of
the following limitations. First, the reading was tested only once. The reading could have
been more accurate if the group had at least two more trials to get the mean and a more
accurate reading. Second, the micropipette may have been used improperly; the group may
have placed too much volume of solution in the cuvette, which would result in inaccuracies.
Third, the dilution of the aliquots when preparing the standard curve may have been
mishandled; to mitigate this, reiterated, the group removed the values of those tubes that
deviated away from the linear regression. Last, the cuvette may have been contaminated,
possibly due to the mishandling of instruments or human error. The cuvette may also not
have been rinsed with ethanol and ddH​2​O properly. contaminants in the cuvette would give
inaccurate results in the spectrophotometer reading. Overall, these limitations all contribute
to the deviation from the actual value.
The limitations of the method itself should also be considered when interpreting the
data. One such limitation is that the Coomassie Brilliant Blue G-250 dye readily binds to
arginyl and lysyl residues of proteins, meaning that it could lead to a variation in the
response of the assay to different proteins. In the original method for the Bradford assay, it
showed large variations in response between different proteins (Walker, 2002) while there
were some adjustments that can be made with the intent of overcoming this limitation such
as adjusting the pH of the solution by adding NaOH to the reagent in order to improve the
assay’s sensitivity and reduce the variation observed in different proteins. However, these
adjustments generally result in a less robust assay that is often more susceptible to
interference by other chemicals (Walker 2002). Another limitation of Coomassie-based
protein assays is their incompatibility with surfactants at concentrations routinely used to
solubilize membrane proteins. Even low concentrations of a surfactant generally causes the
reagent to precipitate. Since the Coomassie dye reagent is highly acidic, a small number of
proteins cannot be assayed with this reagent due to their poor solubility in the acidic reagent
(Pierce Biotechnology, Inc. 2005) (Answer to Q3).
While the Bradford assay has it limitations, it certainly has its advantages that allow it
to be one of the more widely used methods to identify protein concentrations. First, Bradford
assays are generally the fastest and easiest method to perform because they can be done at
room temperature and require no special instruments (Pierce Biotechnology, Inc. 2005).
Second, the Coomassie dye containing protein assays are easily compatible with most salts,
solvents, buffers, thiols, reducing agents, and metal chelating agents that may be
encountered in protein samples (Pierce Biotechnology, Inc. 2005) (Answer to Q3).
On the other hand, other assay techniques utilize the UV range of the
electromagnetic spectrum to acquire results. When ultraviolet radiation is absorbed, it
excites the electrons from the ground state to a higher energy state. Eventually, the
electrons return to their ground state level, and in doing so, release the initial energy they
absorbed as a specific wavelength of light. UV absorption by a chemical compound
produces a distinct spectrum. The theory revolving around this concept states that the
energy from the absorbed ultraviolet radiation is equal to the energy difference between the
higher energy state and the ground state (Choudhary 2017) (Answer to Q4).
Furthermore, it is the Beer-Lambert law that relates the attenuation of light to the
properties of the material through which the light is traveling (Clark 2017). This law states
that whenever a beam of monochromatic light is passed through a solution with an
absorbing substance, the decreasing rate of the radiation intensity along with the thickness
of the absorbing solution is proportional to the concentration of the solution and the incident
radiation (Clark 2017). This law is essentially the combination of two different laws: Beer’s
law which states that the absorbance is directly proportional to the length of the light path, ​l​,
which is equal to the width of the cuvette; and Lambert’s Law which states that the
absorbance is directly proportional to the concentration, ​c, of the solution of the the sample
used in the experiment. Combining both laws we get ​A∝cl which can be made into A= ϵcl. ϵ
is the proportionality constant which is the molar absorptivity or molar extinction coefficient
and is a measure of the probability of the electronic transition.) The equation can then be
written as A=log10(Io/ I)= ϵlc ​(Answer to Q4).
In this experiment, the Coomassie Brilliant Blue G-250 was used to determine the
protein concentration. However, there are also other methods in which we can establish the
relationship between UV absorption and protein concentration without the use of a dye (i.e.
non-colorimetrically). For instance, in spectrophotometry based on UV absorption, protein
concentration can be determined directly by ultraviolet spectroscopy because of the
presence of tyrosine and tryptophan which absorb at 280 nm. Because the levels of these
two amino acids vary greatly from protein to protein, the UV absorbance per milligram
protein is highly variable (Wetlaufer 1962). Another method would be the Biuret reaction. In
alkaline solutions, cupric ions and peptide bonds of proteins and peptides are complexed to
form a purple charge transfer complex (λ​max = 540 nm). The intensity of the color is
proportional to the concentration of protein. Since the number of peptide bonds per given
unit weight is approximately similar for all proteins, this method is reasonably accurate in
general (Gill and Itzhaki 1964). Though its color development time is short and color intensity
remains constant for a reasonable amount of time, it lacks sensitivity, requiring at least 2 mg
of protein, unless it were to use the 310 nm wavelength, which adds further complications of
its own (i.e. many substances in crude protein solutions absorb in that region) (Answer to
Q5).
Analyzing the interactions between biomolecules is important in understanding their
function. Characterizing the binding of proteins to other proteins, to nucleic acids, or to small
molecules is fundamental in biochemical research and finds use in many other areas
including drug discovery (Pol 2010).
For accurate measurement of the interaction kinetics between two interacting
proteins it is essential to know the concentration of specifically binding protein in the
experimental sample used as an analyte (Pol 2010). A spectrophotometer reading of A280
or colorimetric assays (e.g. ones employing Bradford's reagent) is commonly used to
determine total protein concentration (Pol 2010). However, protein impurities can affect the
result. More importantly, both active and inactive forms of the protein are included in the total
protein concentration (Pol 2010). It is important to determine the percentage of specifically
binding protein in the sample, particularly in the case of recombinant proteins, which may be
inactivated due to incorrect folding (Pol 2010).
The measurement of protein concentration in an aqueous sample is an important
assay in biochemistry research and development labs with applications from enzymatic
studies to providing data for biopharmaceutical lot releases (Bailey and Noble 2009).

IV. Conclusions & Recommendations

The group was successful in performing the Bradford assay method to determine the
concentration of protein in a solution. The results of the experiment—particularly the
standard curve and the relationship it shows between the concentration of protein and the
absorbance of the solution at 595 nm—demonstrate the linear relationship between the
aforementioned variables. The group was then able to compute the unknown protein
concentration from its absorbance value as well as the prepared standard curve. By using
the UV-VIS spectrophotometer to obtain the absorbance values, the group was also able to
figure out the role of ultraviolet absorption as well as its underlying principles in determining
protein concentration. From this experiment, the group learned that protein concentration
can be determined through colorimetric means, i.e., with the help of a dye; moreover, the
group realized that determining protein concentration has its relevance and importance in
the fields of medicine and biochemistry such as in the development of pharmaceutical drugs.
To further improve the results of the experiment as well as to mitigate future errors,
the group recommends conducting more trials in getting the absorbance value of the
unknown through the spectrophotometer reading. Doing so would allow the researchers to
obtain the mean and a more accurate reading. Other than that, the group would like to
emphasize on ensuring that the dilution procedures are followed without contaminating the
apparatuses to obtain the best possible standard curve.
V. References
Bailey M, Noble J. 2009. Chapter 8 Quantitation of Protein. Methods in Enzymology:73-95.
[accessed 2018 Sep 23]. https://www.ncbi.nlm.nih.gov/pubmed/19892168
Bradford M. 1976. A Rapid and Sensitive Method for the Quantitation of Microgram
Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical
Biochemistry 72:248-254. [accessed 2018 Sep 23]
Clark J. 2017. The Beer-Lambert Law. Chemistry LibreTexts. [accessed 2018 Sep 23].
https://chem.libretexts.org/Textbook_Maps/Physical_and_Theoretical_Chemistry_Te
xtbook_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Spectr
oscopy/Electronic_Spectroscopy/Electronic_Spectroscopy_Basics/The_Beer-Lamber
t_Law#
Constable E, Housecroft C. 2006. Chemistry: an introduction to organic, inorganic, and
physical chemistry. Harlow: Pearson Education.
Choudhary A. 2017. The principle of Ultra Violet (UV) Spectrophotometer. Medium.
[accessed 2018 Sep 21]. https://medium.com/@ankur1857/principle-of-ultra-violet-uv
-spectrophotometer-e6a1c435d258
Gill D, Itzhaki R. 1964. A micro-biuret method for estimating proteins. Analytical Biochemistry
9:401-410. [accessed 2018 Sep 23]
Khan Academy. 2016. Color Science. Khan Academy. [accessed 2018 Sep 25].
https://www.khanacademy.org/partner-content/pixar/color/color-101/v/color-science-1
Pierce Biotechnology, Inc. 2005. Protein Assay Technical Handbook. Pierce Biotechnology,
Inc.
Pol E. 2010. The Importance of Correct Protein Concentration for Kinetics and Affinity
Determination in Structure-function Analysis. Journal of Visualized Experiments.
[accessed 2018 Sep 23]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3168212/
Sigma-Aldrich. Bovine Serum Albumins. Sigma-Aldrich. [accessed 2018 Sep 23].
https://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?T
ablePage=103994915
The Editors of Encyclopedia Britannica. 2014. Colorimetry. Encyclopedia Britannica.
[accessed 2018 Sep 23]. https://www.britannica.com/science/colorimetry
Walker J. 2002. The Protein Protocols Handbook. 2nd ed. New Jersey: Humana Press Inc.
Wetlaufer D. 1962. Ultraviolet spectra of proteins and amino acids. Adv. Protein Chem
17:303. [accessed 2018 Sep 23]
<WORKING NOTES>
Guide Questions
1. What is the amount of protein (in μg) in your unknown solution?
● As seen in Table 1, the amount of protein in the unknown solution is 36.26 μg.
2. What is the Bradford Assay and how does it determine protein concentration?
● According to Kruger’s “The Bradford Method for Protein Quantitation” article in the
The Protein Protocols Handbook​, The Bradford assay relies on the binding of the dye
Coomassie Blue G250 to protein, as such the levels of the dye detected are
proportional to the amount of protein present.
● Detailed studies indicate that the free dye can exist in four different ionic forms for
which the pKa values are 1.15, 1.82, and 12.4 (2). Of the three charged forms of the
dye that predominate in the acidic assay reagent solution, the more cationic red and
green forms have absorbance maxima at 470 nm and 650 nm, respectively. In
contrast, the more anionic blue form of the dye, which binds to protein, has an
absorbance maximum at 590 nm. Thus, the quantity of protein can be estimated by
determining the amount of dye in the blue ionic form. This is usually achieved by
measuring the absorbance of the solution at 595 nm (Walker 2002)
3. What are the advantages and limitations of the Bradford Assay?
● Advantages
○ Bradford Assays are the fastest and easiest methods of protein assays
because they can be conducted at room temperature and require no special
equipment. (Pierce Biotechnology, Inc. 2005)
○ The Coomassie dye containing protein assays are easily compatible with
most salts, solvents, buffers, thiols, reducing agents, and metal chelating
agents that may be encountered in protein samples. (Pierce Biotechnology,
Inc. 2005)
● Limitations
○ The dye binds most readily to arginyl and lysyl residues of protein meaning
this can lead to a variation in the response of the assay to different proteins.
The original bradford assay showed large variations in response between
different proteins (Walker 2002)
○ There are some adjustments that can be made to attempt overcoming its
limitations such as adjusting the pH by adding NaOH to the reagent in order
to improve the sensitivity of the assay and reduce the variation observed with
different proteins. However, these adjustments generally result in a less
robust assay that is often more susceptible to interference by other chemicals
(Walker 2002)
○ The main disadvantage of Coomassie-based protein assays is their
incompatibility with surfactants at concentrations routinely used to solubilize
membrane proteins. The presence of a surfactant in the sample, even at low
concentrations, generally causes precipitation of the reagent. Since the
Coomassie dye reagent is highly acidic, a small number of proteins cannot be
assayed with this reagent due to their poor solubility in the acidic reagent.
(Pierce Biotechnology, Inc. 2005)
4. Explain the principles behind ultraviolet absorption in solution.
● The absorption of visible light or ultraviolet light by a chemical compound will produce
a distinct spectrum. When ultraviolet radiations are absorbed, this results in the
excitation of the electrons from the ground state towards a higher energy state.
Eventually, the electrons return to their ground state level, and in doing so, release
 the initial energy they absorbed as a specific wavelength of light. The theory
revolving around this concept states that the energy from the absorbed ultraviolet
radiation is actually equal to the energy difference between the higher energy state
and the ground state (Choudhary 2017)
● [Explain the Beer-Lambert Law] ​(Clark 2017)
○ The Beer-Lambert law relates the attenuation of light to the properties of the
material through which the light is traveling.
○ This law states that whenever a beam of monochromatic light is passed
through a solution with an absorbing substance, the decreasing rate of the
radiation intensity along with the thickness of the absorbing solution is actually
proportional to the concentration of the solution and the incident radiation.
○ [since im too braindead rn to type the whole thing]
■ Beer-Lambert’s law is basically the combination of two different laws
namely Beer’s law which states that the absorbance is directly
proportional to the length of the light path ( ​l ), which is equal to the
width of the cuvette AND Lambert’s Law which states that the
absorbance is directly proportional to the concentration ( ​c ) of the
solution of the the sample used in the experiment.
■ Combining both laws we get ​A∝cl which can be made into A= ϵcl (ϵ
being the proportionality constant which is the molar absorptivity or
molar extinction coefficient and is a measure of the probability of the
electronic transition. )
■ The equation can then be written as A=log10(Io/ I)= ϵlc
5. ​Can a relationship between UV absorption and protein concentration be established
non-colorimetrically (without the use of a dye)? How?
● Biuret reaction
○ In alkaline solutions, cupric ions complex with the peptide bonds of proteins
and peptides to form a purple charge transfer complex (λmax = 540 nm). The
intensity of the color is proportional to the protein concentration​. This
reaction occurs only with the peptide bond and not with the amino acid side
chains. Because the number of peptide bonds per given unit weight is
approximately the same for all proteins, this method is generally applicable
and reasonably accurate, irrespective of the composition of the protein
mixture. Other advantages of this method are that the color development time
is relatively short and the color intensity remains constant for a reasonable
amount of time (at least 30 minutes). A major disadvantage of this assay is its
lack of sensitivity, the lower limit being 2 mg of protein. Greater sensitivity can
be achieved by measuring the absorbance of the protein – cupric ion complex
at 310 nm rather than at 540 nm; however, because so many substances
found in crude protein solutions absorb in the near ultraviolet region, this
approach is usually impractical, even when appropriate blanks are included.
Another disadvantage with this assay is that some compounds used in the
laboratory such as Tris buffer
● Spectrophotometry based on UV absorption
○ Protein concentrations can be ​determined directly by ultraviolet
spectroscopy because of the presence of tyrosine and tryptophan which
absorb at 280 nm​. Because the levels of these two amino acids vary greatly
from protein to protein, the UV absorbance per milligram protein is highly
variable.
○ The major advantages of this method include its high sensitivity, ease of
performance, and the fact that the method is nondestructive so valuable
 protein samples can be recovered. Major disadvantages include the
requirement of uv spectrophotometers and quartz cuvettes and the fact that
virtually everything including commonly used buffers absorb in the UV
regions.
●
6. What is the significance of determining protein concentration? What are its applications?