NAME_________________________ DATE___________ PTS ________/10

EXPERIMENT 3: DETERMINATION OF CONCENTRATION OF

PRELAB QUESTIONS:
The following preparatory questions should be answered before coming to lab. They are
intended to introduce you to several ideas that are important to aspects of the experiment. You
must turn in your work to your instructor before you will be allowed to begin the
experiment.

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

2. 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?

3. What size test tubes will you use for the Bradford Assay? Note: (we will be vortexing the
mixture.)

4. How would your results have been affected if you neglected to change wavelengths between
Part A and Part B?

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NAME_______________________________ DATE___________ PTS ________/10

EXPERIMENT 3: DETERMINATION OF CONCENTRATION OF

Adapted from "Experiments in Biochemistry: A hands on approach" by Farrell and Taylor

MATERIALS REQUIRED:

Bradford reagent
Bovine Serum Albumin (BSA)
NADH
Spectrophotometer
Cuvettes
Four Unknowns

Use this space to take notes

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NAME_______________________________ DATE___________ PTS ________/10

EXPERIMENT 3: DETERMINATION OF CONCENTRATION OF

OBJECTIVES:
Upon successful completion of this lab, you will be able to
 Zero the spectrophotometer at a variety of wavelengths and measure the absorbencies of
solutions.
 Decide when dilutions must be made and make the appropriate dilutions.
 Calculate absorbencies, concentrations, and extinction coefficients, using Beer's law.
 Make standard curves and determine concentrations from them.
 Define a reagent blank and decide what reagents must be present in one.
 Design protocals for the creation of a standard curve.

INTRODUCTION:
In this experiment, we deal with the most often used theories and techniques found in a
biochemistry lab, those of spectrophotometry. Along with using a Pipetman, how you use a
spectrophotometer will affect the result of your experiments. If you learn to use them well, your
labs will run smoothly.
Beer-Lambert law:
The abosorbance of light is directly dependent on the concentration of the solution. Bigger the
concentration of the reagents, higher will be the absorbance of the light. The Beer-Lambert law can
be written as
A = cl,
Here
A = absorbance,
 = extinction coefficient
c = concentration of the solution and
l = path length through the solution

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Some points to be considered:
 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 abosorbance, such as
A540 = 0.5.
 The extinction coefficient  is the absorbance of a unit solution concentration and has
units of reciprocal concentration and pathlength. The most common  values recorded are

for a pathlength of 1 cm and 1 M solution. Therefore 600 = 4000 M-1 cm-1 means that a
1M solution has an absorbance at 600 nm of 4000 is a 1 cm diameter cuvett is used.
 Remember that the pathlength l is usually in centimeter and, if not specified, is assumed
to be 1 cm.
 The concentration c has units that are reciprocal of the units for .
 At least 2 mL of the solution is needed in a cuvette in order to read the absorbance.
 Many things can interfere interfere with your use of 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, the light may pass over the solution
instead of going through it. The spectrophotometer must be calibrated before use.
If the substance obeys Beer-Lambert law, then a plot of A versus c is straight. The concentration
versus absorbance curve for solutions of known concentration can be used to determine the
concentration of unknown solution. Beer's law enables you to calculate the concentration of a
substance in solution after measuring the absorbance with a spectrophotometer. Before using a
spectrophotometer, it must be properly calibrated, or zeroed. If it is not, the numbers generated will
be meaningless. Beer's law only works if you know that the relationship between absorbance and
concentration is linear.

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 absence of substance of interest. We handle that by mixing up all
solutions in a tube except that the substance and then read the absorbance. The absorbance of the
reagent blank is then subtracted from the other readings. The blank in this situation is Bradford
reagent. By zeroing the mechine on this tube, the absorbance due to the Bradford reagent is
subtracted automatically.

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Standard curves:
Determining the concentration of a substance using A/l works well if you know the extinction
coefficient () and if you know that the system obeys Beer's law at that concentration. 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 curve.

Colorizing reagent:
Most of the proteins absorbs in ultra-violet range. However, to get quantitative answer, you have
to know the exact  value, which cannot be calculated easily because they depend on the
structure of side chain of amino acids. 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 be measured. Once you have a colored complex, you can use
visible spectroscopy.
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
charge on a protein, it shifts to the blue form, which absorbs maximally at 595 nm. Many
proteins have the same response curve to this dye, making the Bradford method reproducible
among many experiments. 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 up to the same 100-L
volume is not necessary before reagent addition. This saves time in setting up assays.

METHOD:
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 solutions of
NADH, a reagentused later in the course when doing enzyme purification. NADH has an extinction
coefficient of 6220 M-1 cm-1 at 340 nm. The path length for the cuvette is 1 cm.
1. Obtain a solution of NADH of unknown concentration.

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 2. Warm up and zero the spectrophotometer at 340 nm or at the wavelength 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 the dilutions you made to determine the concentration of the NADH
in milimolar (mM).
Part B: Setting up a standard curve
In this part, you set up a standard curve for protein determination, called the Bradford method. The
protein standard is bovine serum albumin (BSA) reagent, 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 in Y axis, we can determine the concentration of unknown proteins from
the graph.
1. warm up the spectrophotometer at 595 nm.
2. Set up ten large, clean test tubes to use for the assays. As a general rule, it is better to use
large test 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 3.1. Using the most accurate pipet available, pipet the BSA
standards into the tubes. The protein concentration is very high, and the volume is low, so
any pipetting error lead to poor standard curves.
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 3 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 absorbancies. Once, the color develops,
it is stable for an hour.
7. Make a plot to determine how much BSA you can add without the curve straing linear. You
may find that it was linear through your higher standard (100 L), but it may also veer off
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 at a lower volume of standard. If the absorbance of the unknown BSA is higher than your
highest standard point that is on the staraight part of the curve, you will need to make up
another tube using less of the unknown BSA. You also want the corrected absorbance of
your unknown to be 0.8 or less.
Protocol for protein determination
Reagent/Tube (mL)
1 2 3 4 5 6 7 8 9
BSA Standard, 0 10 20 30 40 50 75 100 0
1mg/mL (L)
Unknown protein 0 0 0 0 0 0 0 0 ?
Bradford reagent 3 mL 3 mL 3 mL 3 mL 3 mL 3 mL 3 mL 3 mL 3 mL

Waste Disposal:
Have a beaker in the fume hood labeled "Used Bradford Rgt". This should be collected and
handled through hazardous waste disposal. The dye is not particularly toxic to us, but would be bad
for the environment. The solution is fairly strong perchloric acid. Ask your hazardous waste
department how they would like it labeled. It would either be acid or mixed waste.

RESULTS:
Part A -- Beer's Law

1. unknown # ___________________

2. Dilution of NADH ________________

3. Describe how you arrived at this dilution:

4. Absorbance _________________

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Part B -- Bradford Protein Assay

1. Unknown # _______________

2. Fill in the table below for your raw data:

Reagent/Tube (mL) 1 2 3 4 5 6 7 8 unknown

BSA Volume (L)

1 mg/mL

Absorbance

Protein
concentration in μg

ANALYSIS OF RESULTS:

Part A -- Beer's Law

1. Concentration of NADH. ____________________in mg/mL

2. Describe how you arrived at this number:

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Part B -- Bradford Protein Assay

1. Make a proper graph of corrected absorbance vs. g protein and attach it to this report.

2. Calculate the g of protein in your unknown assays.

3. Calculate the concentration of the protein unknown. This is done by dividing the weight of
protein you determined in 2 above by the volume of sample you put into the Bradford
reagent.

Unknown concentration =

POST-LAB QUESTIONS:

1. What is the theoretical absorbance at 340 nm of a 0.01 M solution of NADH, assuming a

2. What dilution would be necessary to get the absorbance from #1 down to 3.1?

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3. Five L of a 10/1 dilution of a sample were added to 5 mL of Bradford reagent. The absorbance
at 595 was 0.78, and according to a standard curve corresponds to 0.015 mg protein on the x-axis.
What is the protein concentration of the original solution?

4. Why did we not use Beer’s law in part B?

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