Experiment 5

Spectrophotometric Determination of
Protein Concentration

Objectives
• Learn how to use a spectrophotometer to calculate concentrations using Beer’s law.
• Calculate absorbances, concentrations, and extinction coefficients, using Beer’s
law.
• Do a simple protein assay and learn how to create standard curves for the
determination of the protein concentration in an unknown.

Background
Almost all biochemical experiments eventually use spectrophotometry to measure
the amount of a substance in solution. Spectrophotometry is the study of the
interaction of electromagnetic radiation with molecules, atoms, or ions.

Light, or electromagnetic radiation, has a wave and particle nature. The wavelength
 of light is the distance between adjacent peaks in the wave. The frequency  is the
number of waves passing a fixed point per unit of time (see Figure 5.1).
 Figure 5.1: Wave nature of light.

These parameters can be further defined by the equation:

=𝒄 where c is the speed of light.
𝒗

Photons of different wavelength have different energies. These energies can be

calculated by the equation:

 = hc/  = h where h is Planck's constant.

Therefore, the longer the wavelength, the less energy the light has and vice versa.

Figure 5.2 shows the relationship between the wavelength of light and the common
types of electromagnetic radiation. As you can see, those regions where the
wavelength is very short correspond to the types of radiation that you know are
powerful and often harmful, such as X-rays, gamma-rays, and ultraviolet radiation.
 Figure 5.2: Wavelength regions of light.

Most compounds have a certain characteristic wavelength or wavelengths of light

that they absorb. Figure 5.3 diagrams this process. Thus, the solution looks green to
us because green light (blue and yellow) is transmitted while the red light is
absorbed.

Figure 5.3: Absorption of light by a solution.

A solution may contain many compounds that absorb at many different wavelengths.
However, if a compound that we are interested in absorbs at a unique wavelength,
we can determine its concentration even in a solution of other compounds.

The Beers-Lambert Law

If a ray of monochromatic light (one wavelength) of initial intensity 10 passes
through a solution, some of the light may be absorbed so that the transmitted light I
is less than 10 (Figure 5.4). The ratio of intensities 1/10 is called the transmittance
and is dependent on several factors:
1. If the concentration c of the absorbing solution increases, then the
transmittance will decrease.
2. If the pathlength l that the light must travel through increases, then the
transmittance will decrease.
3. If the nature of the substance changes or another substance that absorbs more
strongly is used, then the transmittance will change. The nature of the substance is
reflected in , the extinction coefficient, also called the absorptivity constant.

An equation can be written that incorporates these ideas:

Log I0/I is usually called the absorbance and is abbreviated A.

This law A=lc

is called the Beer-Lambert law (Beer’s law).
 Figure 5.4: Relationship between I, I0, l, and c for a solution absorbing monochromatic light.

Some Points to Consider

1. Absorbance A has no units. It is just a number that can be read off of the
spectrophotometer. The wavelength is often specified along with the absorbance,
such as A540 = 0.3.

2. The extinction coefficient  is the absorbance of a unit solution and has units
of reciprocal concentration and pathlength. The most common  values recorded are
for a pathlength of 1 cm and a 1 M solution. Therefore, the expression
600= 4000 M-lcm-1 means that a 1 M solution has an absorbance at 600 nm of 4000
if a 1-cm diameter cuvette is used.

3. Remember that the pathlength l is usually in centimeters and, if not specified,

is assumed to be 1 cm.

4. The concentration c has units that are the reciprocal of the units for .

5. At least 2 mL of solution is needed in a cuvette in order to read it with standard

spectrophotometers.

6. Many things can interfere with your use of a spectrophotometer. If the cuvette
is smudged or scratched, light will be scattered by the tube rather than absorbed by
the solution. If you do not have sufficient volume (see point 5), the light may pass
over the solution instead of going through it. The spectrophotometer must be well
calibrated before use.

If a substance obeys the Beer—Lambert law, then a plot of A versus c is straight, as

in the "ideal" line shown in Figure 5.5. More often, however, the line is curved,
shown as "reality" in Figure 5.5.

Figure 5.5: Absorbance versus concentration.

Reagent Blanks
A reagent blank is a control in which everything is included except the substance
for which we are testing. One problem often encountered in spectrophotometry is
that an absorbance is present at a given wavelength not due to the substance of
interest. We handle that by mixing up all solutions in a tube except that substance
and then read the absorbance. The absorbance of the reagent blank is then subtracted
from the other readings.

Example: We want to read the absorbance at 595 nm of a protein solution mixed

with the colorizing solution called Bradford reagent. What is a suitable reagent
blank?

We want everything that might contribute to an absorbance at 595 nm except the

protein we are trying to measure. Therefore, the best reagent blank is a tube of
Bradford reagent without any added protein. By zeroing the machine on this tube,
the absorbance due to the Bradford reagent is subtracted out automatically.

Standard Curves
Determining the concentration of a substance works well if you know the extinction
coefficient and if you know that the system obeys Beer's law at that concentration
(as in question 1, Pre-lab). When these things are not known, which is most of the
time, a standard curve is prepared. A standard curve is a plot of A versus a varying
amount of a substance. Then, an unknown concentration can be determined from
the graph.

Example: We have a compound X of varying concentrations in a phosphate buffer,

pH 7.0, and the following absorbencies:
Tube no. Concentration Absorbance Corrected Absorbance=
(mM) (Sample Abs -blank Abs)
1 0 0.05 0.00
2 1 0.15 0.10
3 2 0.25 0.20
4 3 0.35 0.30
5 4 0.45 0.40
6 5 0.55 0.50

What is the concentration of a sample of X if the absorbance equals 0.30?

First, you must understand corrected absorbencies. Tube 1 has an absorbance of

0.05, but it does not contain any of the compound that we are measuring. This tube
is our reagent blank, and it has an absorbance. The graph must have a curve that
goes through zero. There are two ways of dealing with this. The first way is to
subtract the absorbance (0.05) from all the absorbencies. This gives the data shown
in the last column. The second way is to zero the spectrophotometer with tube 1.
That way, the subtraction is done automatically. We will always use corrected
curves even though the difference between a corrected curve and an uncorrected one
is largely cosmetic.

Continuing with the example, first subtract the reagent blank (tube 1) from the rest
to give the corrected absorbance; then plot A versus c (corrected) (Figure 5.6). On
the graph, look for the A that corresponds to the unknown, 0.30 - 0.05 (blank) =
0.25. From the graph, A = 0.25 corresponds to the concentration of 2.5 mM, which
is the answer.

Figure 5.6: Corrected absorbance versus concentration for the Example.

Protein Assays
One of the most common uses for spectrophotometry, which also happens to use
standard curves, is the protein assay. Many biochemical studies at some point
require the knowledge of the amount of protein that you have in a sample.
Proteins can be assayed easily if you have a spectrophotometer that can measure
light in the ultraviolet (UV) region. The amino acids tryptophan and tyrosine absorb
strongly at 280 nm, which enables the scientist to scan for proteins at this
wavelength. This is often done as a protein is purified with some chromatographic
technique. However, to get a quantitative answer, you have to know the exact 280
for the protein. If the protein contains few aromatic residues or the extinction
coefficient is low, the UV method would not be suitable. Also, many
spectrophotometers found in teaching labs do not have UV capability.
Many assays can compensate for the inability to use UV absorption. Most of them
depend upon certain dye molecules that react with parts of the protein to give a
colored complex that can then be measured. Once you have a colored complex, you
can use visible light spectrophotometry.
One of the most common and easiest to use is the Bradford method. This method
uses a dye called Coomassie Brilliant Blue G-250, which has a negative charge on
it. The dye normally exists in a red form that absorbs light maximally at 465 nm.
When the dye binds to the positive charges on a protein, it shifts to the blue form,
which absorbs maximally at 595 nm (see Figure 5.7). Many proteins have the same
response curve to this dye, making the Bradford method reproducible among many
experimenters. It is also very rapid. The reaction occurs in a couple of minutes, and
the colored product is stable for over an hour. In addition, the protocol calls for a
protein sample of up to 100-L to be added to 3-5 mL of Bradford reagent. With
such a large difference in volumes between the sample and the protein reagent,
bringing all samples to the same 100-L volume is not necessary before reagent
addition. This saves time in setting up the assays.

Figure 5.7: the principle of Bradford assay.

No single piece of equipment is used more in biochemistry or any life science than
the spectrophotometer. We measure almost everything using one. Granted, most
research labs have very sophisticated spectrophotometers that do more work, but
basically it has a simple light source, a prism or grating for controlling the
wavelength, and a sample holder. Getting caught up in the fun of pushing buttons
without understanding how the machine works is far too easy. A spectrophotometer
with its general principle is shown in Figure 5.8.

Figure 5.8: A spectrophotometer and its general principle.

Apparatus/Reagents Needed

Spectrophotometer
Cuvettes
Micropipettes
Vortex
NADH unknowns Cuvettes
Bradford protein reagent Micropipette
Vortex
BSA (Bovine serum albumin), mg/mL
BSA of unknown concentration
Procedure
Part A: Using Beer's Law to Determine Concentration.
In this part of the experiment, you use Beer's law to determine the concentration of
a solution of NADH. NADH has an extinction coefficient of 6220 M-l cm-1 at 340
nm. The pathlength for the cuvette is 1 cm.
1. Obtain a solution of NADH of unknown concentration.
2. Warm up and zero the spectrophotometer at 340 nm or at the wavelength as
close to 340 nm that you can. Sometimes older machines cannot be zeroed at 340
nm but can be zeroed somewhere between 340 and 360.
3. Make a minimal dilution of the NADH to provide enough solution to measure
in the cuvette.
4. Measure the absorbance of the NADH. If the absorbance is greater than 0.8,
dilute it with water and remeasure. Record these dilutions. You need to know how
much NADH you added to how much water.
5. Use the absorbance and any dilutions you made to determine the
concentration of the NADH millimolar (mM).

Part B: Setting Up a Standard Curve

In this part, you set up a standard curve for a protein determination, called the
Bradford method. The protein standard is bovine serum albumin (BSA), a generic
protein generally used for protein assays. Usually, a series of tubes is set up with
varying amounts of BSA and a constant amount of Bradford reagent. By plotting the
milligrams or micrograms of BSA on the x axis and the corrected absorbance on the
y axis, we can determine the concentration of unknown proteins from the graph.
1. Warm up the spectrophotometer at 595 nm.

2. Set up 10 large, clean test tubes to use for the assays. As a general rule, it is
better to use large tubes for the reactions and then pour a couple milliliters of the
solution into a cuvette-sized tube to read it, rather than setting up the reaction in the
cuvettes. Cuvettes are too small to mix most reaction solutions, and you also risk
permanently discoloring the cuvettes.

3. Set up a protocol as in Table below. Using the most accurate pipet available,
pipet the BSA standard into the tubes. The protein concentration is very high, and
the volume is low, so any pipetting error will lead to poor standard curves.

Reagent /Tube/mL

1 2 3 4 5 6 7 8 9
(Blank) (Unk)

BSA standard, 0 10 20 30 40 50 75 100 -

1 mg/mL (L)

Unknown 0 - - - - - - - ?

Bradford reagent 5 mL 5 5 5 5 5 5 5 5 mL
mL mL mL mL mL mL mL

4. Obtain an unknown BSA solution. Choose a volume of the unknown to assay

and pipet into tube 9. This volume must be 100 L or less.

5. Add 5 mL of Bradford reagent to each tube. Vortex immediately after adding

the reagent to each tube. Do not wait until you have added it to all tubes.

6. Let the tubes sit about 10 min before reading the absorbencies. Once the color
develops, it is stable for over an hour.

7. Make a plot to determine how much BSA you can add without the curve
straying from linear.
Useful links

- Introduction to Protein Structure

https://www.youtube.com/watch?v=HSCUAjZQhXI
- Spectrophotometry and Beer’s Law
https://www.youtube.com/watch?v=zuUvQN8KXOk
- Spectrophotometry- Standard curve
https://www.youtube.com/watch?v=b-mlIWb1lDQ
- Bradford Assay
https://www.youtube.com/watch?v=7o65va089S4
https://www.youtube.com/watch?v=TAMrj0Z9FOk
 Student Name: Student ID no.:
Date: Section:
Instructor Name: TA name:

Experiment 5: Spectrophotometric
Determination of Protein Concentration

Pre-lab Questions
1. We measure the absorbance of a solution of compound X, which absorbs at 540
nm. The cuvette has a width of l cm, the extinction coefficient at 540 nm is 10,000
M-l cm-1, and the absorbance is 0.4. What is the concentration of compound X?

2. If your spectrophotometer can measure an absorbance up to 1.5, what is the

maximum concentration of NADH that you can measure without diluting?

3. What is Vortex? When do use it?

4. When do we use micropipettes not normal graduated pipettes?

5. Convert:
5 mL= L
2 L= mL
2 mL= L
3 L= L
Results and Observations
Part A: Using Beer's Law to Determine Concentration.
- Use the absorbance and any dilutions you made to determine the concentration of
the NADH millimolar (mM).

- If you add 3 mL of water to 1 mL of NADH, mix and get an absorbance of 0.2,

what is the concentration of the original NADH solution? (Show calculations)
(Remember: NADH extinction coefficient = 6220 M-l cm-1 at 340 nm.
MconcVconc = MdilVdil)
Part B: Setting Up a Standard Curve
Suppose you got the following absorbances.

Reagent /Tube/mL

1 2 3 4 5 6 7 8 9
(Blank) (Unk)

BSA standard, 0 10 20 30 40 50 75 100 -

1 mg/mL (L)

Unknown (L) 0 - - - - - - - 80

Final Conc. 0 0.002

(mg/mL)

Absorbance

** One can zero the instrument on the blank or take the blank absorbance and
subtract it from all other readings. Here the instrument was zeroed on the blank.

1- Find all final concentrations in Tubes (3-8).

Example: Tube 2: 10 L (= 10*10-3 mL) were taken from (1 mg/mL) solution and
5-mL Barford reagent was added.

MconcVconc = MdilVdil

1 mg/mL* 10*10-3 mL = Mdil * 5.01 mL

➔ Mdil = 0.002 mg/mL

2- Using Excel draw Absorbance vs. concentration (mg/mL) for Tubes 1-8.
Paste your graph here showing the equation of the Trendline.
(* Don’t forget to set the intercept to zero)

3- From the equation of the graph, find the concentration of the unknown (Tube
9).

4- Remember that the conc. Found above is the final conc. Of the unknown.
Find the original concentration of the unknown.
Post-lab Questions
1. What is the theoretical absorbance at 340 nm of a 0.01 M solution of
NADH, assuming a 1-cm pathlength?

2. What dilution would be necessary to get the absorbance from Question

1 down to 3.1? (consider the final volume to be 2 mL) (Show
calculations).
(-------------L of 0.01 M NADH to ------------ L water)

3. Absorbance at 340 nm of a 0.02 mM NADH solution is 0.124 with a 1-

cm pathlength. What is the absorbance with a 1.2-cm pathlength?

4. Five L of an unknown BSA sample were added to 5 mL of Bradford

reagent. The absorbance at 595 nm was 0.78 and, according to a standard
curve, corresponds to 0.015 mg/mL of protein on the x axis. What is the
protein concentration of the original solution?

5. Why did we not use Beer's law in Part B?

6. Why is the absorbance versus concentration curve for a substance rarely

straight for all concentrations? (i.e. deviation from Beer’s law).