CSUSM BIOL 210 Lab Introduction to Cellular and Molecular Biology

Biology
Procedures:
Be sure to make note of ALL modifications to the lab procedures in your lab notebook for full credit!
Check off the steps as you complete them.

Part A: Protein Extraction

Overview of Protein Extraction

Obtain and cut up a fruit or vegetable sample into small pieces

â
Use liquid nitrogen and mortar and pestle to homogenize the sample into a fine powder
â
Lyse the cells and stabilize the proteins in extraction buffer
â
Centrifuge the sample at high speed, which will separate the sample into a pellet that
contains cellular debris (which you do not want) and a supernatant that contains the
extracted proteins (which you DO want)

1. ¨ Select one fruit or vegetable that you would expect to have high PPO activity (i.e. a fruit or
vegetable that rapidly turns brown when cut and exposed to air).

2. ¨ Use a razor blade to cut the sample into the size of a small pea, and place it on a piece of
weigh paper or a weigh boat. Take the mass of your sample, and record the mass in your lab
notebook. What will you use as the tare for your mass measurement?

3. ¨ Quickly chop the material into very small pieces, and then place the pieces into a mortar.

4. ¨ Prepare and label the 50 mL conical tube containing extraction buffer with your group name.
Prepare and label three 1.5 mL microfuge tubes with your group name and numbers 1, 2, or 3.

5. ¨ Your instructor will pour liquid nitrogen into the mortar to freeze the tissue. Also, cool the
head of the pestle in liquid nitrogen.

a. Safety precautions: Liquid nitrogen is extremely cold, maintaining a temperature of

negative 196 °C (-196 °C). It is dangerous if not handled with extreme caution. Wear
gloves at all times, and be careful not to expose your skin directly to the liquid nitrogen
as it will burn your skin like molten lava.
b. For this experiment, the low temperature of liquid nitrogen will crack the cells open for
us (just like it would to your own body cells) and keep the sample cold. This makes the
difficult process of grinding the sample easier and minimizes unwanted enzyme activity
in the protein extract.

6. ¨ Thoroughly crush the tissue into a fine powder (like dry flour) with the pestle. The tissue
must not thaw during this process. Ask your instructor for more liquid nitrogen if necessary.

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CSUSM BIOL 210 Lab Introduction to Cellular and Molecular Biology
Biology
You will need to make sure all the plant cells are mechanically broken apart (homogenized) so
you can release as much of the total protein as possible from of your sample.

7. ¨ Use a metal spatula to scrape the frozen tissue powder into the labeled 50 mL tube
containing 12 mL of ice-cold extraction buffer and 0.5 g of insoluble polyvinylpyrrolidone (PVP).

a. Extraction buffer contains: 50 mM Tris base (pH 8.3), 11% glycerol, 1% PEG 8000,
0.015% citric acid monohydrate, 0.010% cysteine monohydrate, and 0.010% ascorbic
acid. The extraction buffer helps maintain a specific pH and contains several
antioxidants. This mixture, along with cold temperatures, stabilizes the total protein
extract so that the PPO proteins will still function when we measure enzyme activity in a
future lab.
b. PVP can form complexes with phenolics and alkaloids. The complex formation allows
removal of phenolics and alkaloids from plant samples, thereby inhibiting any
modifications of proteins by them and any hindrance they may cause in
spectrophotometric determinations of protein content. PVP might also enhance the
stability of enzymes (Sigma Aldrich).

8. ¨ Vortex the tube for 30 seconds.

9. ¨ Place your sample tube into the table-top centrifuge. Note: Prior to starting the
centrifugation cycle, check to be sure that your tubes are properly balanced. Your instructor will
show you the correct positioning of the tubes in the rotor.

10. ¨ Spin your cells for 10 minutes at 3500 rpm at 4 °C.

• After centrifugation (because of centrifugal force), you will see a solid pellet collected at the
bottom of the tube and liquid supernatant separated above it. You may also see a lipid pad
at the very top.
• Your total protein extract will be in the supernatant. It is important to remember that the
pellet and the lipid pad can contaminate your protein extract and interfere with subsequent
steps.
• The pellet contains the "heavier" components of the cell, like the cell walls, organelles and
fragments of cell membranes. Sometimes the pellet is what a researcher is looking for. This
time, it is not. For this experiment, the components of the pellet will interfere with your
results.
• The lipid pad may also be present. This is a floating "raft" of fats (known as lipids). This layer
does not contain PPO proteins, and may contaminate your sample. Therefore, it should also
be carefully avoided.
• Only the supernatant contains a mixture of the soluble molecules that you will need for the
Bradford assay and the next two labs. The supernatant contains many cell macromolecules
but predominantly proteins (by mass). The supernatant also includes (but is not limited to!)
the PPO enzyme you will be investigating in a future lab with an enzyme activity assay.

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CSUSM BIOL 210 Lab Introduction to Cellular and Molecular Biology
Biology
11. ¨ Carefully transfer 1 mL of the supernatant to EACH of your three labeled 1.5 mL microfuge
tubes using a pipette.

• Depending on the quality of your sample and centrifugation, you may find it easier to use a
P200 tip, P1000 tip, or P1000 tip that you have cut the end off (2-3 mm) with scissors.
• What is most essential is to only take the supernatant, avoiding the pellet and lipid pad! It is
better to have quality rather than quantity!

12. ¨ Tube 1 will be used, as described below, for Bradford analysis. Place it in your ice bucket.
Tube 2 will be used for SDS-PAGE next week. Tube 3 will be used for the enzyme assay the
following week. Store tubes 2 and 3 in the ice bucket by your instructor’s desk. These samples
will be stored at -20 °C.

Part B: Protein Quantitation (Bradford Assay)

Overview of Protein Quantitation

Perform the calculations, then generate a set of BSA samples of known protein
concentration
â
Perform the calculations, then generate dilutions of your protein extract of unknown
protein concentration
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Add Coomassie dye to all samples and take spectrophotometer readings (absorbance at
595 nm wavelength of light)
â
Generate a standard “curve” using your BSA samples
â
Use the best-fit equation to calculate the protein concentration of your protein extract

A number of very simple and inexpensive spectrophotometric methods are used to measure total
protein. These include assays like Bradford, Lowry, UV, and others. In this lab, you will use one of the
most commonly employed tools in biochemistry research labs today: the Bradford assay.

The Bradford assay depends on Coomassie Brilliant Blue G-250 Dye (Figure 1). Often called Coomassie,
the molecules that make up this dye will bind to several different amino acids: arginine, phenylalanine,
tyrosine, histidine, and tryptophan. Once bound to one of these amino acids, the absorbance of the
Coomassie “dye” molecule changes from 465 nm (free Coomassie) to 595 nm (Coomassie bound in a
complex), which can be detected using a spectrophotometer (Figure 2). Thus, in the presence of
Coomassie, the result of an increase in total protein present in a sample also results in an increase in
the 595 nm absorbance reading (Abs595).

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CSUSM BIOL 210 Lab Introduction to Cellular and Molecular Biology
Biology
Figure 1: Chemical structure of Coomassie Brilliant Blue G-250 Dye (Thermo Fisher)

Figure 2: Detection of Coomassie Brilliant Blue G-250 Dye using a spectrophotometer

Again, the amount of bound Coomassie dye is proportional to the protein concentration of the
solution. Thus, we can use the predictability of Coomassie binding, in conjunction with analysis of
samples with known concentrations, to determine the concentration of unknown samples.

Your Abs465 vs Abs595 results are based on the absorbance and transmittance (It in Figure 2) of light at
these two particular wavelengths (each selected, in turn, by the wavelength selector). Because the
absorbance and transmittance of light is directly proportional to the concentration of protein in a
solution, thanks to the presence of Coomassie Blue, a standard curve can be generated.

Figure 3 shows a standard “curve” based on dilutions of a known protein, bovine serum albumin (BSA),
of known concentrations. Scientists use this linear plot of “absorbance versus known protein
concentration” as a molecular “ruler” to measure the concentration of unknown samples. During this
lab, you will prepare dilutions of BSA with known concentrations, record your spectrophotometer
results of these BSA samples as well as your total protein extract samples (with unknown
concentrations). After this lab, you will use these results to plot your data using Microsoft Excel (or

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CSUSM BIOL 210 Lab Introduction to Cellular and Molecular Biology
Biology
another spreadsheet program) to generate a scatter-plot and best-fit equation. You can think of this
equation as the molecular “ruler” to measure the protein concentration for your total protein extracts.

Figure 3: Plot of Absorbance at 595 nm (OD) vs. BSA concentration (ug/mL)

BSA concentration (ug/mL)

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CSUSM BIOL 210 Lab Introduction to Cellular and Molecular Biology
Biology
Figure 4: Overview of Bradford Assay to determine protein concentration. Note that this example
describes adding 20 uL of a series of protein standards, but you will calculate the appropriate volumes
according to the lab manual procedures.

13. ¨ Preparing your standard curve: You will be provided with a BSA stock solution of known
concentration (1 mg/mL BSA). What is 1 mg/mL BSA converted to units of ug/mL? In your lab
notebook, clearly calculate all the volumes needed to make your serial dilutions in the table
below. Complete this table before the start of this week’s lab.

• Example: Given your BSA standard, how would you prepare 0.5 mL (or 500 uL) of a BSA
solution whose concentration is 4 ug/mL?
• Recall: C1V1 = C2V2
• Concentration of BSA stock (C1) = 1 mg/mL = 1000 ug/mL
• C2 and V2 are the concentration and volume you want to prepare. V1 is what you want to
solve for.
• (1000 ug/mL)*V1 = (4 ug/mL)*(500 uL)
• V1 = 2 uL
o Note that this is the volume needed of the stock solution.
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CSUSM BIOL 210 Lab Introduction to Cellular and Molecular Biology
Biology
o Check that your units cancel when you are performing the algebra. Otherwise, you
need to perform unit conversions!

• To achieve the desired final volume of 500 uL, you will need to add the appropriate amount of
water/solvent: 500 uL – 2 uL BSA stock = 498 uL water

Preparation of samples for BSA standard curve

Tube label BSA Total volume Volume of BSA Volume of water
Concentration (uL) stock (uL) (uL)
(ug/mL)
0 500 0 500
5
10
15
20
25
Preparation of samples of unknown concentration
Tube label Dilution Total volume Volume of Volume of water
(uL) protein extract (uL)
(uL)
1:10 500
1:100 500
1:400 500

14. ¨ Prepare and label all the 1.5 mL microfuge tubes you will need to complete this exercise.
How many tubes will you have?

15. ¨ Preparing your diluted protein extracts: In order to ensure that the Abs595 readings of your
samples fall within the linear range of the standard curve, you must dilute your sample. In your
lab notebook, clearly calculate all the volumes needed to make your diluted protein extracts in
the table above. Complete this table before the start of this week’s lab.

16. ¨ Pipette the volumes that you calculated in the table above to prepare your BSA standards
and diluted protein extracts in the labeled 1.5 mL microfuge tubes. Store your tubes on ice
while you prepare all of them.

17. ¨ Move all of your tubes to a microfuge tube rack. To each tube, add the equivalent volume
(500 uL) of Coomassie dye. Vortex each tube briefly to mix.

18. ¨ Incubate all nine tubes at room temperature for 5 minutes. While you are waiting, label nine
plastic disposable cuvettes with the sample name. Make sure you write the label at the top of
the cuvette, out of the pathway that light will travel in the spectrophotometer.

19. ¨ Transfer the 1 mL solution from each microfuge tube into the appropriately labeled cuvette.
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CSUSM BIOL 210 Lab Introduction to Cellular and Molecular Biology
Biology

20. ¨ Take a picture of your samples in the cuvettes. Print out your photo after lab, and tape it
into your lab notebook (black and white is fine). Label your photo, and describe what you see in
your lab notebook.

21. ¨ Determine the Abs595 for each sample, using the same spectrophotometer for all of your
readings. There will be nine scans total: one blank, five BSA standards, and three diluted protein
extracts. Try to work as efficiently as possible since the blue color may darken over time. Which
sample will you use as your “blank” and why?

22. ¨ Record all of the concentrations and corresponding Abs595 values in a data table in your lab
notebook.

23. ¨ Make sure to document any other observations that stood out to you in your lab notebook
under the Results section.

Part C: Protein Quantitation (Bradford Assay) Data Analysis

24. ¨ Use Microsoft Excel (or another spreadsheet program) to generate a scatter plot with BSA
concentration on the x-axis and Absorbance at 595 nm (Abs595) on the y-axis. Be sure to
properly label the axes on the graph, and include an appropriate title for your graph. Plot only
the Abs595 values of your BSA standards. Do not plot the absorbance values for the three diluted
protein samples!

25. ¨ Insert a linear trendline (standard curve), including the equation of the line and R2 value on
your graph.

26. ¨ Print out a copy of this graph, and tape it into your lab notebook under the Results section.

27. ¨ Using the equation of your standard curve, calculate the protein concentration in your
protein extract. You must show your work/calculations in your lab notebook for full lab
notebook credit! Note that your extracts were diluted prior to the assay, so you must account
for the dilution factor to determine the protein concentration of the original (undiluted)
extract. For example, if you calculate that the 1:10 dilution of the protein extract has a
concentration of 50 ug/mL, what would the concentration of the undiluted protein be? Would
it be 5 ug/mL or 500 ug/mL?

a. You have Abs595 values for three dilutions of your protein extract. In theory, you could
calculate the protein concentration using all three Abs595 values, but they would likely
lead to different values. What dilution of protein extract will you choose to determine
your protein concentration? Why did you choose this value? Write the answers to these
last two questions in the Results section of your lab notebook.

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