Experiment 4: Protein Assay Spectrophotometry

Experiment 4:
Protein Assay
Spectrophotometry
Theory
• Spectrophotometry is the study

electromagnetic radiation emitted or
absorbed by a given chemical species.
• A Spectrophotometer is an instrument
that measures the amount of light
absorbed by a substance.
UV-Vis Spectrophotometer
The major components of the UV-vis spectrophotometer
Incident Emergent
Watch the following video to see the major components light light
of the spectrophotometer and how it works. cuvette
https://www.youtube.com/watch?v=BST5GRsAnPk
The sample will absorb
certain amount of light
→ I0 ≠ I
Beer's Law:
I0 : intensity of the incident light.

I: intensity of the light after it has passed through the sample.
l: distance travelled by light through the sample = pathlength of the cuvette, usually =1cm
Absorbance (A): is the amount of light absorbed by the sample.
Beer's Law states that the concentration of a chemical solution is directly proportional
to its absorption of light.
A = log I0/I = εlc A is unitless.
ε :absorptivity/ absorption coefficient/ extinction coefficient (M-1.cm-1)
c: concentration (M or mol/L)
Think of Beer‘s law as a straight-line equation
If you have 5 solutions of known
concentration (we call them standard
solutions) and one with an unknown
concentration. What to do to know the
unknown concentration?
1. zero the machine on the blank

solution (a solution without the
material to be tested).
2. measure A of the standard
solutions.
3. Plot A vs. Concentration = standard
curve
4. Calculate the slope of the obtained
straight line.
5. Slope = ε x l, l = 1 cm → ε can be
calculated.
6. Measure the A of the unknown →
apply Beer’s law with the known
slope you have → you got the
unknown concentration.
Practice problem
We measure the absorbance of a solution of compound X which absorbs light

at a wavelength of 540 nm. The cuvette has a width of 1 cm, the extinction
coefficient at 540 nm is 10 000 M-1.cm-1 and the absotbance is 0.4. What is the
concentration of compound X?
A = εlc
0.4 = 10 000 x 1 x c
→ c = 4 x 10 -5 M
Experiment: Protein Assay
Objective: Determination of the unknown concentration of protein in a solution by Bradford
method. The protein standard Bovine Serum Albumin (BSA) will be used for this assay.
one of the most common uses of spectrophotometry is the protein assay. Many biochemical
studies at some point require the knowledge of the amount of protein that you have in a
sample.
Colorizing reagent : in order to use visible –light spectrophotometer, your protein must present
in a colored complex form using certain dyes that react with parts of the protein to give this
complex. In this experiment we will use Bradford method.
This method uses a dye called Coomassie Brilliant Blue G-250, which has –ve charge on it, it has
a red color and absorb the light maximally at 465 nm. When the dye bind to the +ve part of the
protein, it shifts to a blue form that absorbs maximally at 595 nm. This rxn is rapid that takes
only 2-3 min to produce that color which is stable for almost an hour.
Please watch: https://www.youtube.com/watch?v=7CTYSBkCNQQ
• A standard curve (calibration curve) is a plot of A vs. a varying amount of a substance.
This plot should be linear, and can be fit with the equation y=mx+b. The equation of the
best-fit line can then be used to determine the concentration of the sample, by using the
instrument signal to correlate to concentration.
• Target:
1. construct a a calibration curve by measuring the absorbance of a series of solution
(standard solutions) containing varying amounts of the compound to be assayed.
2. From the calibration curve you can find the extinction coefficient and then use it to
calculate the concentration of the unknown samples.
Please watch the following to see how to perform the experiment.

In this video they bought the standard solution.. They can be also prepared in the lab.
https://www.youtube.com/watch?v=vfY3mVOlGBU
Practice problem
Protein assay by Bradfored method
Firstly a spectrophotometer was warmed up at Y= 37.5x
595 nm then it was zeroed at the blank
solution. After that, Absorbances of the
standard solutions were taken. The following
calibration curve was then plotted.
If the spectrophotometer gave A= 0.6 for the
protein solution of the unknown concentration.
Determine the protein concentration in the
unknown sample. Protein concentration (x 10-3 M)
You have 2 methods to get the unknown conc. 2. From the staright-line eqauation of the plot.
1. Directly from the plot. A = εlc → y= 37.5 x , l= 1 cm, ε= 37.5 M-1.cm-1

check the X value of when Y = 0.6 A= 37.5 c
X is the conc. = 16 x 10-3 M 0.6 = 37.5 c
c = 16 x 10-3 M

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Experiment 4:

• Spectrophotometry is the study

I0 : intensity of the incident light.

1. zero the machine on the blank

We measure the absorbance of a solution of compound X which absorbs light

Please watch the following to see how to perform the experiment.

1. Directly from the plot. A = εlc → y= 37.5 x , l= 1 cm, ε= 37.5 M-1.cm-1

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