BCH 372

Modern Concepts in Biochemistry Laboratory

Laboratory 1
Biochemistry Boot Camp, Spectrophotometry
and BSA Standard Curve
The purpose of the first part of the laboratory session is to review some of the basic
laboratory techniques and calculations that will be needed for this semester's lab work. As part
of this lab, you will learn to use graduated cylinders and glass pipets correctly and to read
laboratory balances accurately. You will also review the use of micropipetters and practice using
them with a simple spectrophotometric method. Finally, you will do several types of basic
biochemical calculations.
The purpose of the second part of the laboratory is to learn how to use a
spectrophotometer to calculate concentrations using Beer’s law. You will also do a simple
protein assay to validate pipetting skills and create standard curves for the determination of the
protein concentration in an unknown.

I. Pre-Lab Preparation

Be sure to purchase a lab manual and a lab notebook and bring them to the lab along
with your calculator and the other lab supplies noted in the syllabus. Download and print a copy
of the Data Sheet for Laboratory 1. While you should record your data in a laboratory notebook
first, you will eventually need to fill out the Data Sheet and turn it in next week. Before the lab,
please read Chapter 1 in the lab manual Experiments in Biochemistry: a hands-on approach,
second edition, by S. O. Farrell and L. E. Taylor. Focus on sections, 1.3, 1.4, 1.5, 1.6, 1.7 and
1.9.

Before the lab, please read Chapter 3 in the lab manual Experiments in Biochemistry: a
hands-on approach by S. O. Farrell and L. E. Taylor. Be sure you understand the basic
properties of light, the Beer- Lambert Law, and the purpose of constructing a standard curve.
Focus on sections 3.1 and 3.2 of Chapter 3 of the lab manual on spectrophotometry. Look
closely at the calculations shown in Practice Sessions 3.1, 3.2, and 3.3. Notice that there are
several ways of preparing standard curves, which are discussed on pages 70-72. We will use
Method 2 in which the response (absorbance) is plotted as a function of the amount of the
substance of interest. This method simplifies the calculations. It does require, however, that the
final volume in all of the assay tubes be the same. This is normally very easy to do.

Please note that on pages 12-13 in Chapter 1 of the lab manual, the authors describe a
dilution factor in terms of the ratio of the final volume or concentration over the initial volume
or concentration. For example, if 10 ml of a solution is diluted to a final volume of 100 ml, the
dilutions factor would be defined as:
 Df = 100 ml/10 ml = 10.

This is a somewhat unusual convention. In most laboratories and in the laboratory handouts to
be used in this course, the more common convention of defining a dilution factor as the ratio of
the initial volume or concentration to the final volume or concentration will be used.

Df = 10 ml/100 ml = 1/10 or 10-1 dilution.

Special note:
Dilutions: dilution factor is the ratio of the initial volume or concentration to the final
volume or concentration.
Df = 10 ml/100 ml = 1/10 or 10-1 dilution.

II. Laboratory Procedures Part 1

A. Use of Graduated Cylinders and Pipets

While micropipetters are extremely useful for transferring small volumes of liquid and
will be frequently used in this class, there are times when it is necessary to transfer a volume of
liquid greater than 1.0 ml (1000 μl). Graduated cylinders are commonly used for volumes of 10
to 1000 ml and glass pipets are used for volumes of 1 to 25 ml. The purpose of this part of the
experiment is to learn to use these materials accurately.

1. Graduated Cylinders

For relatively large volumes of liquid (greater than 10 ml), graduated cylinders are most
often used. A graduated cylinder has markings on the side of the glass or plastic which indicate
the volume. Depending on the total capacity of the cylinder, the meaning of the markings will
vary. For a 10 ml, 25 ml, or 50 ml cylinder, the markings are most often 1 ml apart. For a 250
ml or 500 ml cylinder, they are most often 10 ml apart. One of the problems with most cylinders
is that an aqueous solution (that is, one made up in water) often shows a pronounced meniscus or
curvature when it is placed in the cylinder. This is due to the binding of water molecules to one
another and to the sides of the cylinder. As shown in the next figure, the volume in a cylinder is
read most accurately by looking at it straight on and reading the volume at the bottom of the
meniscus. A graduated cylinder can be used either to measure the volume of unknown solution
or to measure out a certain volume of solution and then to transfer it to another container.
2. Pipets

For intermediate volumes of liquid (between 1 ml and 25 ml), pipets are most often used.
A pipet is a calibrated piece of glass or plastic tubing with markings along the side. Again,
depending on the total capacity of the pipet, the meaning of the markings will vary. For a 1 ml
pipet, the markings may be either 0.1 ml or 0.01 ml apart. For a 5 ml or 10 ml pipet, the
markings are usually 0.1 ml apart. Each pipet usually is marked at the top so that the gradations
are clear: it will say 5 in 1/10 ml or 1 in 1/100 ml. As with graduated cylinders, there is often a
pronounced meniscus with aqueous solutions, so it is important to hold it vertically when making
a measurement. A certain volume of liquid may be transferred from one container to another by
drawing the liquid up into the pipet and then allowing a volume of liquid between two specific
markings to flow out. Mohr or measuring pipets are calibrated only to a point near the bottom of
the glass or plastic tube. A specific volume is delivered by measuring the amount of liquid
between two markings. Serological pipets are calibrated all the way to the tip and so must be
"blown out" in order to deliver all of their volume. This is illustrated in the following figure,
where the top pipet is a Mohr pipet, and the lower pipet is a serological pipet.

To use a pipet, it is necessary to draw liquid up into it. In the past, this was often done by
sucking on the end of the tube with your mouth (as with a straw) and covering the end with your
index finger. However, this type of mouth pipetting is no longer considered safe and various
types of bulbs, pipet aids or pumps, or propipets are used instead. MOUTH PIPETTING IS
NOT ALLOWED IN THE COURSE. In this class, plastic pipet pumps will be used as shown in
the following figure.
 A green pump should be used with 5 ml or 10 ml pipets; a blue pump should be used with
1 ml pipets. To use this type of pump, insert the top end of the pipet into the pump and twist it
gently so that it seals against the plastic. Then rotate the plastic wheel to draw the liquid up into
the pipet. To let liquid out of the pipet, rotate the wheel in the opposite direction. It is helpful to
touch the tip to inside of the container into which the liquid is to be placed so that it flows out
evenly.

B. Measuring Volumes with a Graduated Cylinder and Pipet

1. Take a 100 or 150 ml beaker and fill it about half full with the red colored liquid in a
flask at your station. The liquid is just water with some food coloring added to it so you
can see the solution easily. Note that while the stock flask or the beaker may have
markings on their sides, these values are only approximate and should never to be used
for measurement. Pour the liquid into a 100 ml graduated cylinder. Look at the
meniscus, measure the volume, and record the results below.

volume of red liquid: _______ ml

2. To test the accuracy of this measurement, place a clean dry beaker on one of the top-
loading balances. Press the TARE button to reset it to 0.00 g. Pour the liquid into the
beaker and measure the weight in grams. The density of pure water is 1.00, so 1.0 ml =
1.0 g. Record the weight of liquid below:

weight of red liquid: ________ g

Note that at one atmosphere of pressure, the terms weight and mass can be used
interchangeably.

3. Now fit a 10 ml serological pipet with a green pipet pump. Draw some of the red liquid
in the flask up into the pipet so that it is above the 0 ml (or 10.0 ml) mark. Look at it
carefully. Be sure that the pipet is sealed onto the pipet pump and the liquid does not drip
out the tip. Rotate the wheel and then transfer 10.0 ml of liquid into the original flask.

4. Repeat this process by practicing with volumes of 6.7, 5.8, 4.2, and 3.1 ml. Note that you
can use the markings anywhere along the pipet to transfer a certain volume. You can also
read the pipet from the top down or from the bottom up.
5. Once you have figured out how to read the markings correctly, place a clean dry beaker
on the top-loading balance and reset it to 0.00. Using the pipet, transfer 7.3 ml of liquid
to the beaker and measure the weight in g. The transfer will be most accurate if you wipe
the outside of the pipet with a Kim-Wipe and touch the tip of the pipet to the glass and
allow the liquid to run into the container. Push out as much of the liquid as possible.
Record the weight and reset the balance to 0.00. Repeat the process four more times and
record your values in your lab notebook using the following headings. Then calculate
with average (mean), the % error, and the mean deviation as described in section 1.4 and
on pages 31-32 of the lab manual.

6. Now repeat the process by doing five trials with a volume of 3.8 ml. Again, calculate
with average (mean), the % error, and the mean deviation as described in section 1.4 and
on pages 31-32 of the lab manual.

trial 7.3 ml 3.8 ml

1 ________ ________

2 ________ ________

3 ________ ________

4 ________ ________

5 ________ ________

average (mean) ________ ________

% error ________ ________

mean deviation ________ ________

C. Use of Micropipetters

The objective of this part of the experiment is to review the basic procedures for the use
of micropipetters. These instruments will be used throughout the semester and will be critical to
the experiments.

1. Each group will be provided with a set of three micropipetters: a P-20, a P-200, and a P-
1000. Boxes of small and large sterile plastic tips also will be provided.
2. Review the structure of each instrument which is illustrated below.

3. Note how the volumes are indicated. For a P-20, three numbers are usually shown. The
black numbers represent the volume in microliters (µl) and the red number the volume in
tenth of a microliter. For a P-200, three numbers are usually shown which represent the
volume in microliters. For a P-1000, all four numbers are sometimes shown. However,
often only three numbers are shown, which means that the last digit is missing.
Consequently, 1 0 0 indicates 1000 µl, but 0 1 0 indicates 100 µl. This is shown in the
next figure.

There are many different brands of micropipetters, and before using any micropipetter,
you should look at the display and see how it is set up. Also, you should turn the thumb
 wheel carefully to the volume desired. Never turn the thumb wheel above or before the
minimum and maximum volume for a particular micropipetter.

4. Remember that you must always use a tip with a micropipetter. Put a small plastic tip on
a P-20 or P-200 micropipetter and large plastic tip on a P-1000 micropipetter.

5. To transfer a particular volume of liquid, depress the plunger just to the first stop and
draw the liquid up slowly. Check to see that here are no air bubbles in the liquid
in the tip. This is commonly a problem if you just “pop” the plunger up or if you are
using a P-1000 micropipetter. Wipe the outside of the pipet tip with Kim-Wipe to
remove any extra liquid. Then place the tip into the container or solution to which you
want to add the liquid to and depress the plunger all the way down to the second stop. If
you are combining two solutions, draw the liquid up and down several times to
rinse out the tip and mix the solutions together. Then eject the tip into the
designated container.

III. Laboratory Procedures Part 2

A. Spectrophotometry
Recall from Cell Biology that there are four basic methods for detecting a particular type
of molecule: 1) spectrophotometric assays, 2) radiochemical assays, 3) activity assays, and 4)
immunological assays.
1. Spectrophotometric assays. This type of assay involves exposing molecules to light
and then measuring either the resulting absorption or fluorescence. Light absorption or
fluorescence may result directly from the intrinsic chemical properties of the molecules
of interest. Alternatively, they may occur indirectly as a result of treating the molecules
of interest with other compounds which react with them to create new chemicals that
exhibit absorption or fluorescence.

2. Radiochemical assays. This type of assay is based on the incorporation of a radioactive

isotope such as 3H, 14C, 32P, or 35S into the molecules of interest. The molecules then can
be detected or traced through the release of energy in the form of beta-particles (high-
energy electrons) or gamma-radiation.

3. Activity assays. This type of assay involves measuring the activity or effect of the
molecules of interest. Most enzymes are detected by their ability to catalyze a specific
chemical reaction and most transport proteins are detected by their ability to bind a
substrate or to translocate it across a cellular membrane. In these cases, the amount of the
molecule of interest (the enzyme or binding protein) is inferred from the amounts of other
molecules (for example, an enzyme's substrates or products).

4. Immunological assays. This type of assay is based on the use of antibody proteins that
bind specifically to the molecules of interest. Binding of antibodies may lead to the
formation of a visible precipitate, a radioactive complex, or an enzyme-linked complex
that can catalyze a chemical reaction.
 Of these assays, spectrophotometric assays are usually the simplest and the most
convenient to perform. In the case of an enzyme, the amount of activity is usually inferred from
the rate at which one of the substrate(s) disappears or one of the products(s) is formed. This can
be inferred from a change in absorbance or fluorescence at a particular wavelength.

To assess the accuracy of your pipetting, you will measure the absorbance of each tube in a
Spectronic 20 Genesys spectrophotometer at 600 nm. These are the same instruments you have
used in Cell Biology and other courses on the West campus. The basic components are shown
below.

The instrument should be used in the following way:

a. Be sure the power cord is plugged into a grounded 120 Volt outlet.
 b. Turn on the power switch on the back of the instrument. The instrument will go
through a short power-up sequence that takes about 2 minutes. Then allow the
instrument to warm up for 15 minutes before taking any readings.

c. Press the A/T/C button on the key pad to select absorbance.

d. Press the nm (UP) or nm (DOWN) buttons on the key pad to select the
wavelength to be used (595nm).

e. To set the instrument to zero absorbance, lift up the cover of the sample
compartment and insert test tube or a plastic disposable cuvette containing the
reference solution appropriate to your experiment. Be sure to wipe the outside of
the cuvette first with a Kim-Wipe and insert the cuvette correctly so that
light passes through the clear walls. Note the arrow in the sample compartment
that indicates light passes from the back of the instrument towards the front.
Close the cover of the sample compartment.

f. Press the 0 ABS/100% T button on the keypad to set the instrument to 0

absorbance. The zero reading will appear on the LCD display.

g. To make an absorbance reading, remove the test tube or cuvette with the reference
solution and insert another test tube or cuvette containing the sample solution.
Again, be sure the outside of the cuvette is clean and is oriented correctly.
.
h The absorbance of the solution will appear on the LCD display. Record the
number in your lab notebook.

i. Once the instrument has been standardized, it should remain stable and you can
continue to take readings without resetting the instrument to 0 absorbance.

B. Preparation of a Protein Standard Curve

The purpose of this part of the experiment is to prepare a protein standard curve, using
bovine serum albumin (BSA) as the protein standard and the Bradford (Coomassie Blue)
Reagent.

1. You will be provided with a 1.0 mg/ml stock solution of bovine serum albumin (BSA).
To improve the accuracy of the standard curve, you will 1) add water to each tube to
ensure that the total sample volume is 100 μl; 2) do all of the assays in duplicate; 3)
carefully wipe the outside of the pipet tips with a Kim-Wipe; and 4) invert rather than
vortexing the solutions with the Bradford Reagent.

2. Set up 17 13 x 100 mm glass tubes as shown in the following table.

tube water 1.0 mg/ml BSA Bradford Reagent

1 100 μl 0 μl 3.0 ml
 2 95 5 3.0
3 95 5 3.0
4 90 10 3.0
5 90 10 3.0
6 85 15 3.0
7 85 15 3.0
8 80 20 3.0
9 80 20 3.0
10 70 30 3.0
11 70 30 3.0
12 60 40 3.0
13 60 40 3.0
14 50 50 3.0
15 50 50 3.0
16 40 60 3.0
17 40 60 3.0

*Note that tubes 2 and 3 are duplicates, tubes 4 and 5 are duplicates, and so on.
Using duplicate samples will allow you to make a more accurate standard curve.

3. Using micropipetters, add the water to the tubes first. Then add the BSA solution. It will
help the accuracy if you use a new tip for each sample and wipe off the outside of the tip
quickly with a Kim-Wipe. Also, when you add the BSA, put the tip into the water and
draw the liquid up and down several times to rinse the inside of the tip and mix the water
and BSA together.

4. When all of the samples have been prepared, add 3.0 ml of Bradford Reagent to each
tube using the Repipetter (Inside Fume Hood).

5. Cover each tube with part of a square of Parafilm and invert it several times. This is
better than vortexing the samples because it does not generate a lot of foam.

6. Allow the tubes to sit at room temperature for 10 minutes.

7. Measure the absorbance of each tube at 595 nm. The tubes will fit directly into the
sample compartment of the spectrophotometer Use tube # 1 to set the instrument to zero
absorbance since this "blank" contains only water and Bradford Reagent. Calculate the
average absorbance value for each of the duplicate samples.

8. Calculate the average absorbance value for each of the duplicate samples. Then plot the
average absorbance values as a function of the amount of BSA in each tube (Y axis –
absorbance and X axis – amount). Note that because the concentration of the standard is
1.0 mg/ml, 1 μl contains 1 μg of protein. Draw a "best fit" line through data points. It
should go through the origin (0 BSA = 0 Absorbance) and be linear through at least some
of the points. You might find, however, that the standard curve becomes nonlinear at
high protein concentrations. Make your graph using Excel.