BIO 330 Cell Biology
Laboratory
Spring 2011
Measuring Protein and DNA in Biological Samples
Measurements are often made of certain protein levels or mRNA levels in biological cell, tissue, or organ
samples. However, in order to account for the amount of starting material, which can vary greatly, the
end results are usually “normalized” per amount of total protein, total RNA, or total DNA. For example,
in Dr. DuPriest’s research on the enzyme chymase, her results were expressed in terms of enzyme
activity per g protein used in the initial reaction.
An additional concept required for measurement of many proteins and/or nucleic acids is the concept of
the standard curve. A standard curve is something a researcher constructs using increasing KNOWN
amounts of material (protein or DNA). Then, after figuring out an equation that matches the data
obtained from the standards, we can use the standard curve to figure out the concentration of our
UNKNOWN samples.
In this activity, we will prepare a crude extract of beef liver. We will then measure the concentrations
and amounts of protein and of DNA in that extract. To do that, we will need to construct standard
curves of pure protein (bovine serum albumin, BSA) and pure DNA. Both our samples and standards will
be read by spectrophotometry according to the protocol given below.
Protocol
1. The whole class will prepare a single homogenate of beef liver using the blender; each pair of
students will receive an aliquot of the homogenate to work with. It is important to keep the liver and
homogenate on ice at all times, except when you MUST remove it from ice for a specific reason. The
class should obtain a large piece of liver. Using a kitchen knife and cutting board, cut the liver into
cubes, and measure out about ~50g (+/- 5 g). At the same time that some people are weighing and
cutting the liver, others should measure out the appropriate amount of homogenization buffer.
(Homogenization buffer contains NaCl to match salt levels in tissue; detergent to split open cells; pH
buffer to keep pH around 7.4 and some other minor components.) The amount of buffer should
approximately match 10X the weight of liver. (E.g., ~ 500 mL of buffer for 50g of liver). The buffer
should be kept on ice until ready for homogenization. When both liver and buffer are ready, combine
them in the pre-chilled blender. Put the lid on securely, and start the blender on medium level for 5-10
seconds, then switch to high power for two more 10-second bursts. Pour into a pre-chilled Erlenmeyer
flask and place in the refrigerator for 20 minutes, swirling to mix approximately every five minutes. This
time allows the proteins and DNA to move from the tissue into the aqueous buffer.
2. Pour the homogenate into six pre-chilled 50-mL centrifuge tubes; fill the tubes ~ 2/3 full. The tubes
must be weighed to ensure they are balanced. Eyeball the one that appears to be fullest; weigh it first
and record its weight. Then measure the next ones in succession, adding more homogenate until it
weighs the same as the first one. Place the tubes on ice while you are measuring the others. Take the
samples downstairs to the chemistry lab, to the large centrifuge. Load the centrifuge tubes into the
rotor. Centrifuge at ~1500xg for 10 minutes. Carefully remove the tubes, place back on ice, and bring
BIO 330 Cell Biology
Laboratory
Spring 2011
them back upstairs, being careful not to jar the samples and remix them. (Make sure to turn the
centrifuge off when you are finished.)
3. Transfer the supernatant (top layer now cleared of debris) to a new tube (you can leave some of the
supernatant in the tube to avoid disturbing the pellet). The pellet is now waste material and can be
discarded. The supernatant will now be referred to as your liver extract. This type of extract can be
used in a wide variety of applications, such as measuring enzyme activity, or to measure specific protein
levels. In this activity, we will simply measure the concentrations of protein and DNA in the extracts.
However, before we do that, we must prepare our standards.
4. Prepare your protein standards. You will be making a four-point standard curve, plus a blank. You
will start with the most concentrated standard (the stock solution) first, then make serial dilutions to
obtain your other concentration. Record the concentration of the most concentrated standard in your
notebook. You should start a table where you will record protein concentration and the
spectrophometer readings you will get later. (See the last page of this handout for an example.)
Transfer 250 L of the stock solution to another tube, and add 250 L of homogenization buffer to the
tube. Calculate the concentration of this solution, and mix thoroughly. Then take 250 L of that
solution, transfer to another tube, and add 250 L of homogenization buffer and mix as before. Again,
calculate the concentration. Repeat this again to make a total of four solutions, including the stock
solution. Also transfer some homogenization buffer into a similar tube to use for your blank. Label each
tube clearly with the concentration you have calculated (or 0 for your blank) – keep track of the
appropriate units. Set aside.
5. Prepare your DNA standards. Again, you will make a four-point standard curve, plus a blank.
Measure out 1 g purified DNA, and dissolve in 10 mL homogenization buffer. Dissolve completely, then
transfer 5 mL to another tube. Add 5 mL of homogenization buffer, mix thoroughly. Repeat two more
times to make a total of 4 tubes. Again, make a blank tube. Store these tubes on ice or in the
refrigerator. Calculate concentrations and label as for protein standards. Also, make a separate table in
your notebook for DNA concentration vs spectrophotometer reading. DNA concentrations can be
measured as-is; no assay is necessary. Go to Step 7 if your group is measuring DNA first. If your group is
measuring protein first, go to Step 6.
6. Protein Concentration Assay and Spectrophotometry. To measure protein concentration in a
biological sample, we perform a Bradford assay in which we combine our liver extract with a dye that
changes color when it binds with certain amino acids present in every protein. Follow these steps:
Obtain two plastic rectangular spectrophotometer cuvettes for each concentration of your
standards, plus two for your sample (total of 12). Transfer 2385 uL of water to each cuvette. Add 15 uL
of your protein standard, sample, or blank. You cannot label these cuvettes, but keep them lined up so
you are sure of which one is which. Once all 12 cuvettes are prepared to this point, quickly add 600 uL
of Bio-Rad Protein Assay Reagent to each tube, then carefully mix each sample: use a piece of Parafilm
to cover each cuvette, seal using your index finger and invert 3-4 times to mix. Try to complete all of
these within about 2 minutes. Let all the reactions incubate at room temperature for 15-30 minutes,
BIO 330 Cell Biology
Laboratory
Spring 2011
then read on the spectrophotometer. You should be reading the absorbance at 595 nm, which
corresponds to the blue color that develops when the red dye combines with protein. You should be
able to see that the protein standards that contain high amounts of protein contain more blue color.
Follow instructions laid out by the laptop spec station to take your measurements, and record your data
in your notebook. Be sure to record your sample reading on a separate line, and leave space to
calculate what the concentration and amount of protein is in your sample. To do this, you will need to
use Excel to construct your standard curve, putting your averaged protein concentration on the x-axis
and the A595 values on the y-axis. The resulting plot should be linear. After you plot your points (exclude
your blank for this step), right-click on any point, and select Insert Trendline. Make sure to select the
options to show r-squared value and show equation on chart. Using the equation (you will need to
rearrange it using algebra), plug in your averaged A595 value for your liver extract as y and solve for x to
determine the concentration of protein in your liver extract. This concentration will be in the same units
as you calculated for your standards.
7. DNA Spectrophotometry. To measure nucleic acid concentration in a biological sample, we simply
measure the absorbance at 260nm. Typically in research, we do not construct a standard curve,
because results nearly always come out as expected…we can simply multiply our spectrophotometer
reading by a constant. However, because we are using crude extracts instead of more sophisticated
extracts, we will use the standard curve method. Blank the spectrophometer first by transferring the
contents of your blank tube to a quartz spectrophotometer cuvette…follow the instructions located near
the laptop UV spec station. After blanking the spec, measure each of your standards. Measure each
sample twice, recording the A260 and A280 value for each reading. Construct a standard curve as
described in Step 6 for protein, using your averaged A260 values. Calculate the concentration of DNA in
your liver extract as described for protein. To assess the purity of your samples, for each standard and
for your liver extract, calculate a ratio of A260 to A280 (calculate a ratio for each reading, then average
your duplicate ratios). Typically, we expect to see a purity ratio of 1.6-1.9 – your standards should fall in
this range. However, since we are using crude liver extracts, we expect that your ratio will be lower
than 1.6.
Conclusion/Discussion
In your discussion, summarize what you concluded about today’s lab activity, and explain how a
researcher uses a standard curve to determine the concentration of an unknown sample.
BIO 330 Cell Biology
Laboratory
Spring 2011
Example Table
Protein Concentration
(mg/mL)
Replicate 1
Replicate 2
Average
100
50
25
0.975
0.554
0.368
0.982
0.552
0.372
0.9785
0.553
0.370
12.5
0.227
0.218
0.2225
Example Graph
Protein Assay Standard Curve
1.2
A595 value
1
y = 0.0085x + 0.1348
R² = 0.9972
0.8
0.6
0.4
0.2
0
0
20
40
60
80
Protein Concentration, ug/uL
100
120