Biophotonics Lab: Quantitative Protein Analysis
Biophotonics is general term for all techniques that deal with the interaction between biological
items and photons. This refers to emission, detection, absorption, reflection, modification, and creation of
radiation from biomolecular, cells, tissues, organisms and biomaterials. Areas of application are life science,
medicine, agriculture, and environmental science. This has become a very useful procedure in
biotechnology as it allows for an accurate measurement of the relative amounts of molecules present in a
solution.
In this specific lab, we will be measuring the amount of protein in a substance using two methods;
Relative quantification and Absolute quantification. Both methods will utilize reagents that will bind to
proteins and allow us to measure the amount of reaction. In either method, generally the more reactivity of
a reagent, the more protein present in the solution.
Part A: Relative Quantification
In this experiment we will be using a Biuret Reagent Test. The Biuret reagent is made of
potassium hydroxide (KOH) and copper (II) sulfate (CuSO 4), together with potassium sodium tartrate
(KNaC4H4O6·4H2O). The blue reagent turns violet in the presence of proteins, and changes to pink when
combined with short-chain polypeptides. The greater the quantity of protein, the more violet the solution
will be.
Procedures:
1) Obtain a test tube holder and 5-7 test tubes
2) Aliquot 1ml of the solutions into the test tubes so the each tube has a different
solution. Remember to use a different pipette for each solution to prevent
cross contamination.
3) Record the solutions and test tubes in the chart below.
4) Add 1ml of the Biuret reagent into each tube and gently swirl the contents.
Wait one minute for the reaction to fully catalyze.
5) Using your best judgment, rank the level of reaction starting with 1 being the
most reactive. Record in the table below.
Test Tubes
Solution
Rank
Part B: Absolute Quantification (Beer’s Law)
In this experiment we will be performing a Bradford protein assay. The Bradford assay, is a
colorimetric protein assay, is based on an absorbance shift in the dye Coomassie. The Coomassie reagent
changes in solution and binds to proteins. Binding of the proteins stabilizes the blue form of Coomassie dye,
thus the amount of complex present in solution is a measure for the protein concentration by use of an
absorbance reading.
In order to perform this test we must use a spectrophotometer. A spectrophotometer is a device
that measures the amount of light (electromagnetic radiation) a solution (sample) absorbs. We will be using
a simple spectrophotometer called a colorimeter to measure the amount of light your samples absorb. A
sample put into the path of an electromagnetic ray absorb a quantity of energy from that ray and transmits
the remaining energy. The amount of transmitted energy is always less than the amount of incident energy.
In short, as light is sent through the sample and absorbed by the Coomassie-protein complex, the remaining
light will transmit through to the detector at a fraction of the original amount of light. This is called
transmittance, but by measuring this amount, we can infer the amount of absorbance of the sample and
therefore the amount of protein in a sample, this is called Beer’s Law.
Procedures:
1) Follow the instructions given to calibrate your colorimeter.
2) Obtain enough curvettes for each sample you will be testing, and add 1ml of
the Bradford reagent to each.
3) Add 20ul from the dilute solutions (50 parts dH20 to 1 part sample) to
separate curvettes. Remember to use the micropipette and change the tips for
each solution.
4) Cap the curvettes with the caps and flick the side of the curvettes several times
to mix. Let stand for 1-2 minutes.
5) Starting with the known concentration solutions (standards), begin placing
each in the colorimeter to obtain an absorbance percentage. Record this
information in the table below.
6) Conclude the testing with the unknown samples and record the information in
the data table.
Curvettes
Solution
%A
g/ml
7) For the unknowns, we do not yet know what the g/ml is, so we must develop a
standard curve using our standards. But our concentrations and quantities are
so small we need to adjust them so that we can expand the graph. So, from
each g/ml value for the standards, multiply that value by 100. This will give
us a value per 100ml not 1ml.
8) Plot this information (%A and g/100ml) on the graph below.
9) Draw a best fit line through the points starting at the point f origin (See
example).
10) Now, find the g/ml of the unknowns on the best fit line by corresponding the
absorbance level (y axis) with the g/ml (x axis). Record the values in the data
chart.
EXAMPLE:
Analysis Questions:
1. Which part of this lab was Qualitative and which was Quantitative? Explain
your answer.
2. Compare the results of Part A to Part B. Did your rank in Part A match the
data obtain in Part B? Explain this comparison and cite evidence to support.
3. Reflecting back on this lab, which method is the most accurate? In what
circumstances would each of these methods be appropriate to determine
protein concentrations?