BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS
LAB 3
PAGE DETERMINATION OF PROTEIN FINGERPRINTS
STUDENT GUIDE
Information from an NSF sponsored workshop was used in adapting this lab: Genomics: from Mendel to Microchips, taught by the
Partnership for Plant Genomics Education at the University of California Davis, July 2005. The lab was originally adapted from
Biotechnology Explorer ™ Protein Fingerprinting Instruction Manual Bio-Rad DNA Fingerprinting – Partnership for
Research and Education in Plants.
GOAL
The goal of this lab is to separate proteins by polyacrylamide gel electrophoresis and to
analyze the data using a standard curve graphed using MS Excel.
OBJECTIVES
After completion, the student should be able to
1. Perform a common type of plant protein extraction
2. Perform the technique of SDS-PAGE.
3. State the purpose for SDS-PAGE in the research laboratory.
4. Explain how proteins will move through SDS polyacrylamide gels when placed in an
electric field.
5. Explain the need for denaturation and reduction when separating proteins by size.
6. Estimate the molecular weight of a protein using proteins of known molecular
weights as standards.
7. Analyze electrophoresis data using manual and Excel graphing.
TIMELINE
This lab will take 2 laboratory periods:
DAY 1: Prep for the lab, extract plant proteins, run SDS PAGE, stain and destain
DAY 2: Graph results using MS Excel and analyze
BACKGROUND
Proteomics is the study of the complete set of proteins present in an organism. The
number of proteins in an organism far exceeds the number of genes, which is due in part
to molecular mechanisms of gene control. These processes include shuffling of DNA to
form different genes (such as that seen in antibody production), post transcriptional
control, and post translational control of protein production. Proteomics is a growing field
that employs sophisticated molecular techniques such as two dimensional gel
electrophoresis and mass spectrometry. Understanding the basis of protein separation by
polyacrylamide gel electrophoresis is the first step in understanding proteomics.
Electrophoresis is a technique that separates charged molecules in an electric field.
Negatively charged molecules migrate in an electric field toward the anode; positively
charged molecules move toward the cathode. In polyacrylamide gel electrophoresis
(PAGE), molecules move through a porous gel matrix that separates molecules on the
basis of both charge and size. This migration is complicated because both the size (mass)
and the net charge of the molecule contribute to the migration. Proteins can have different
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BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS
charges because the side chains of many amino acids carry a charge. Proteins move
through the gel based on their charge to mass ratio. That is, the higher the negative
charge, the faster the protein migrates; conversely, the larger the size, the slower the
protein migrates. The separation of proteins in polyacrylamide gels on the basis of their
charge to mass ratio is called non-denaturing or native gel electrophoresis. This technique
is most commonly used when the native conformation and activity of the protein must be
maintained.
Many proteins of different sizes have similar charge to mass ratios; these proteins would
migrate very similarly in a polyacrylamide gel, and therefore would not be resolved.
Another problem with native gel electrophoresis is that many proteins consist of multiple
subunits that make the molecule too large to separate easily by polyacrylamide gel
electrophoresis. These problems can be overcome by another technique called denaturing
gel electrophoresis, or SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this
technique, proteins are treated with a strong anionic (negatively charged) detergent called
sodium dodecyl sulfate (SDS) that binds to proteins in proportion to the size (mass) of the
protein (about one molecule of SDS per amino acid). That is, a protein of 20 kD would
bind twice as much SDS as a protein of 10 kD. In addition to the influence of mass on
protein movement through polyacrylamide, protein shape can also affect the migration
rate. To separate two proteins strictly on the basis of size, they must also have the same
shape. When protein samples are denatured by heating and treatment with reducing
agents such as -mercaptoethanol (that break disulfide bonds between two cysteine
amino acids), the polypeptides that make up the protein separate, unfold, bind SDS and
assume a rod-like structure. Since the length of these rod-like molecules is proportional to
the size of the polypeptide, denatured SDS polypeptides migrate in polyacrylamide gels
primarily on the basis of size. Proteins separated by SDS-PAGE can be compared to
denatured polypeptides of known size to determine their mass.
Polyacrylamide is a synthetic polymer or chain of acrylamide monomers. These
acrylamide chains can be crosslinked to each other by the addition of bisacrylamide
during the polymerization reaction. The bisacrylamide crosslinks cause the chains to form
a mesh-like structure, in which the holes of the mesh represent the pores that retard
protein migration in the gel. At higher acrylamide or bisacrylamide concentrations, the
mesh becomes tighter with smaller pores that more strongly retard the migration of
proteins. Proteins can be separated on polyacrylamide gels of a consistent concentration
determined by the sizes of the proteins to be separated. Large proteins require a lower
concentration of acrylamide in the gel. Acrylamide concentrations in the range of 10% to
15% separate proteins of about 12,000 to 70,000 daltons. In many instances, separation of
proteins can be improved by the addition of another layer of acrylamide of a different pH
atop the separating gel. This top layer, called a stacking gel, has large pores that allow
rapid migration of the proteins until they reach the boundary where the separating gel
begins. The protein migration abruptly slows, and the proteins stack, so that they enter as
a thin zone at the surface of the separating gel. The pH and concentration differences
between the two gels result in well defined, narrow protein bands that are better resolved
making analysis easier. A gradient gel is used to separate proteins of widely different
sizes while also separating those of similar sizes. The highest concentration, which
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retards small proteins, is at the bottom of the gel and the lowest concentration, for larger
proteins, is near the top.
In addition to the concentration of acrylamide in gels, the buffer system affects the
migration of the proteins. A discontinuous buffer system, in which the buffer and the
gel have dissimilar ions, helps to stack the proteins at the boundary of the separating gel.
After the proteins stack, they migrate through the separating gel with a constant pH and
voltage, allowing resolution by the sieving action of the acrylamide pores, thus separating
the proteins by size.
Separated proteins can be visualized by staining with Coomassie stain, a blue dye that
binds strongly to proteins. Before the proteins can be stained, they must be bound or
fixed to the gel matrix with acetic acid. The coating of SDS must also be removed from
the proteins so they are accessible to the Coomassie blue dye. SDS is removed from the
gel and the proteins with methanol or isopropanol. The Coomassie stain contains
methanol, acetic acid and Coomassie blue. After the proteins are stained, the gel must be
"destained" so the protein bands become visible. The destain solution contains lower
percentages of methanol and acetic acid. Coomassie blue can detect as little as 0.1 g of
protein per band.
In this laboratory SDS-PAGE is used to analyze plant protein samples. The proteins will
be resolved on a precast gel (check the manufacturer for the concentration) using a
discontinuous buffer system. The molecular weights of the major plant proteins isolated
by the class will be calculated based on their migration relative to the protein standards of
known sizes. The proteins of the molecular weight standards are prestained with
conjugated dye. Protein samples are mixed with a loading buffer/dye solution that
contains SDS and -mercaptoethanol to denature the proteins. Sucrose or glycerol is
included in the loading buffer to increase the density of the sample so it can be loaded on
the gel, and bromophenol blue is included to help visualize the progress of migration
during electrophoresis. Most vertical gel chambers accommodate two gels at the same
time. In this case, 2 teams will work together to prepare samples and run two gels
simultaneously on one gel chamber. Three unknown samples will be run with molecular
weight protein markers.
LABORATORY OVERVIEW
In this lab, young plants of various types will be used to extract proteins from different
developmental regions, i.e., leaves, stem, flower, sepals and roots. The crude protein
extracts will be run on an SDS polyacrylamide gel, which will be stained, destained and
analyzed for different patterns. Prominent protein bands will be sized using a standard
curve, and conclusions will be drawn regarding the possible identification of these
proteins.
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SAFETY GUIDELINES
The electric current in a gel electrophoresis chamber can be dangerous. Always turn
off the power supply before removing the lid or touching the gel. Make sure that the
counter where the gel is being run is dry.
MATERIALS:
Per class
Bio-Sage Coomassie Blue stain (destains with dH2O)
Practice gel loading solution
10X Tris-glycine-SDS buffer stock
Vortex mixers
Heating block set at 95°C
1000 mL graduated cylinder
dH2O
1 L Corning orange capped bottles
Plastic wrap
White light box
Polaroid camera and film
A supply of transfer pipettes
Labeling tape
1.5 ml microfuge tubes
Per every 2 teams
Flat metal spatula for separating gels
Dual vertical mini gel rig with clamps
Power supply
Per team
Kaleidoscope prestained protein molecular weight markers – aliquot 25 μl per team (BioRad # 161-0324) NOTE: heat briefly at 37°C to dissolve any precipitated SDS before aliquoting.
Flowering plants, one/team
2x Protein loading dye/buffer (≥ 100 ul)
Scalpel with sharp blade
Precast 15% polyacrylamide gel for SDS PAGE
8 disposable pellet pestles
2 ml 1X Laemmli buffer
1000 l micropipetter and tips
20 l micropipetter and tips
Metric ruler
Scissors
Sharpie marker
Small beaker of 10-15 1.5 ml microtubes
Microtube rack
Plastic dish for transport of gel
Plastic staining dish
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BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS
RECIPES
10x Tris-Glycine-SDS Buffer (500 ml should be allowed for each double gel rig; 1x =
25 mM Tris; 192 mM glycine, 0.1 % SDS )
3.04 g Tris base
14.41 g glycine
1.0 g SDS
dH2O to give 100 ml final volume
Dilute 100ml in 900ml dH2O for 1x working concentration
10 x Laemmli Buffer
0.25 M Tris,
1.92 M Glycine
1 % SDS in aqueous solution
Dilute to 1x working concentration
2x Protein loading dye (10 ml)
1.2 ml 1M Tris HCl pH 8*
4 ml 10% SDS
2 ml 100% glycerol
1 mg bromophenol blue
0.1 ml -mercaptoethanol
2.7 ml dH2O to give 10 ml final volume
*Adjust to pH 8 with HCl for prepoured graduated gels; if using discontinuous self poured
gels, adjust pH to 6.8.
PROCEDURE
Part I. Prep
Teams 1 and 2: Set two heating blocks with 1.5 ml tube blocks at 95°C. Gather supplies
needed and distribute to all teams. Set up an ice bucket for each team.
Teams 3 and 4: Prepare 2 liters of 1x Tris-Glycine-SDS electrophoresis buffer from the
laboratory stock. (Note: Two liters is enough for three gel rigs/6 gels.) Verify your
calculation with your instructor before diluting. Label the bottle of buffer per cGMP.
Part II. Isolating plant proteins
1. Label 6 or 7 1.5 ml microcentrifuge tubes with your team number and the plant
tissue type.
2. Use a sharp scalpel to cut the plant tissues to obtain the equivalent of the size of
two Tic-Tac breath mints. Be sure to take the following samples: stem, flower,
sepals, roots, top leaves, and bottom leaves. If seeds are available in the lab, they
can also be used. Place each tissue into its labeled tube.
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Petals
Sepals
BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS
3. Grind each tissue with its own pellet pestle for no less than 5 minutes. Do not
cross contaminate samples – use a different pestle for each!
4. After each tissue is ground, add 250 μl of 1x Laemmli buffer and grind for
another 3 minutes.
5. Incubate each sample for 5 minutes at room temperature.
6. Add 30 μl of 2x protein loading dye to each sample and flick to mix.
7. Heat the team samples for five minutes at 95 C. Spin for 2 minutes to pellet the
plant debris and place on ice until ready to load.
Part III. Running and Staining the Gel
The instructor will demonstrate how to set up a vertical gel for SDS-PAGE.
1. Prepare the gel by removing the comb and the piece of tape at the bottom. Clip the gel
to the vertical electrophoresis chamber with the short glass plate next to the gasket on
the top buffer reservoir. Add running buffer to cover the wells in the top reservoir and
enough in the bottom reservoir to immerse the slit in the glass plate exposed when the
tape was removed.
2. Any debris and bubbles should be removed from each well by using a transfer pipette
to gently flush the wells with running buffer.
3. Check the information that accompanied the gels to verify well capacity, as this may
vary depending on the gel manufacturer. Load the gel in the following order. (NOTE:
loading dye should be diluted 1/10 if it is too thick.)
Lane 1. 10 μl 2x protein loading dye
Lane 2. 10 μl molecular weight protein markers
Lane 3. 10 μl 2x loading dye
Lane 4. 20 μl sample – top leaves
Lane 5. 20 μl sample – bottom leaves
Lane 6. 20 μl sample – sepals
Lane 7. 20 μl sample – stem
Lane 8. 20 μl sample – flower
Lane 9. 20 μl sample – seeds (or 10μl 2x protein loading dye)
Lane 10. 10 μl 2x protein loading dye
(NOTE: vertical polyacrylamide gels sometimes run unevenly due to unequal
distribution of the heat generated during running. To help prevent „smiling‟ of the
samples, no wells should be left empty.)
Gel showing
„smiling.‟
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BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS
4. Run the gels at 125 volts until the bromophenol blue dye front migrates to the end of
the gels, which takes about 45 minutes. Do not let the dye front run off the gel.
5. Add 50 ml Coomassie stain to a small plastic container.
6. When the gel is finished running, turn off the power supply and remove the lid.
Gently pry the glass plates apart using a spatula. Submerge the plate, gel-side-down,
into the stain to allow it to release from the glass.
7. Place the lid on the container and shake gently for 30 minutes.
8. Pour off the stain and add 100 ml of destaining solution. Shake gently for 30
minutes. Change the destain solution, and continue shaking until the protein bands
are visible. The gel can be left in destain overnight or longer.
9. Place the destained gel on a white light box and take a photo. The Polaroid camera
should have a yellow filter, and an f-stop setting of 22 - 32. An exposure time of
1/125 second may give good results but this will depend on each light box. For digital
photos, use the white light illuminator and the hood from the Polaroid lab camera to
obtain the correct distance above the gel.
DATA ANALYSIS
Part I. Manual Graphing
The proteins will appear as blue bands. The size of the most abundant proteins can be
determined by setting up a standard curve using the size of the molecular weight markers
and the distance each marker band migrated from the well. Measure the distance (in
millimeters) that each band of protein migrated from the bottom of the well to the middle
of each band and record in the data table. Measure the distance that the dye front
migrated in each lane and record it in the table. Calculate the relative mobility (Rf) for
each protein by dividing the distance migrated by the distance the dye front migrated. Set
up a data table to include the distance each protein migrated, the distance the dye front
migrated at the bottom of each lane, the Rf value for each, and the molecular weight of
each protein. Use semi-log graph paper to graph the standard curve by placing the
molecular weight of each marker on the Y (ordinate) axis of the graph and the Rf value
on the X (abscissa) axis. See a sample data table and sample graph in Table 1 and Figure
1, below. Include your data table and this graph in your notebook as part of your results.
(NOTE: the log of the molecular weight size of each standard protein should also be
calculated and added to the table for use in Part II.)
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BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS
Table 1. Data Table of SDS PAGE Results
Standards & Unknowns
Distance of
Migration
Distance
Dye Front
Migrated in
this lane
Rf
(X axis)
Molecular
Weight in
daltons*
(Y axis)
log mol. Wt.
(for use in
Part II)
Myosin (blue)
Beta-galactosidase
(magenta)
BSA (green)
Carbonic anhydrase (violet)
Soybean trypsin inhibitor
(orange)
Lysozyme (red)
Aprotinin (blue)
Unknown protein 1
Unknown protein 2
Unknown protein 3
*The dye attached to each of the standard proteins causes each to run a little more slowly. These sizes are
not the actual size but are corrected for the dye content on each. Kaleidoscope Markers have different
molecular weights for each lot – CHECK THE DATA SHEET ENCLOSED WITH THE MARKERS.
Figure 1. Example of a Standard
Curve for Protein Molecular
Weight Determination
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BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS
Part II. Graphing a Standard Curve using Excel.
Graphing with Excel is more accurate than a manually drawn graph. By using the
formula for the slope of the line,
y = mx + b
(slope = m; y intercept = b)
Excel formulas can be used to automatically calculate the size of unknown proteins also
run on the same gel. Set up Data Table 1 in an Excel spreadsheet. Use Excel to graph the
standard curve by placing the log of the molecular weight of each marker on the Y
(ordinate) axis of the graph and the Rf value on the X (abscissa) axis. Use the formula for
a line and Excel functions to determine the sizes of the unknown proteins on the gel. Do
not round off any calculations; use all six places to the right of the decimal. The function
to calculate the log is Log10. For anti-log calculations, select the “Power” function and
use 10 for “number” and the log for “power.” Print your data table with the embedded
graph and include it in your results. The table and graph must include appropriate labels
and legends. A sample Excel data table and graph are given, below.
SDS-PAGE Determination of Unknown Protein Size
Distance
Distance dye front
of
migrated
migration
(mm)
(mm)
23
29
35.5
43
27
32
36
y=mx+b
45
45
45
45
45
46
45
m=slope
Rf (X
axis)
Log Mol. Molecular
Wt. (y)
Weight
0.51
4.97
0.64
4.83
0.79
4.63
0.96
4.48
0.60 4.866449
0.70 4.755449
0.80 4.644449
-1.11
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ID
94000 MWt Marker
"
67000
"
43000
"
30000
73,527 Unknown B
56,944 Unknown C
44,101 Unknown D
intercept = b
5.5324491
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BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS
QUESTIONS
1. What were the sizes, based on your graphs, of the major proteins observed? Compare
these data with that in other groups to determine if there are any patterns that emerge,
i.e., did all plants have the same major protein bands for the same plant structure
(e.g., leaves)?
2. Use the Internet to find the kilodalton sizes of some abundant plant proteins, e.g.,
rubisco (enzyme responsible for fixing carbon). Articles can be found on the class
Blackboard website that may help get you started.
3. Explain the action of -mercaptoethanol and SDS in preparation of proteins for SDS
PAGE.
4. Explain the difference between native polyacrylamide gel electrophoresis and
denaturing PAGE.
5. A protein migrates on SDS-PAGE at the same speed as the 80,000 Dalton molecular
weight marker fragment. When the protein is further analyzed using other
instrumentation, it is found to have a mass of 65,000 Daltons not including the
carbohydrate moiety covalently attached to it. Assuming that the latter is the correct
size of the protein, why did it indicate a mass of 80,000 Daltons using SDS-PAGE?
6. IgG contains 2 small and 2 large polypeptide chains. A preparation of IgG was
incubated with SDS, heated and electrophoresed using SDS polyacrylamide. One
major band near the top of the gel was observed after staining. Explain these results.
7. Compare and contrast the results from your semi-log hand drawn graph and your
Excel graph.
8. The size of DNA fragments that have migrated in an agarose gel can also be
determined by using a standard graph of the molecular weight marker fragments.
Since DNA fragments normally run uniformly across an agarose gel, the Rf is not
used, but rather the distance the fragments actually migrated in the gel. Complete the
Excel Graphing Practice on the next page. Email your Excel spreadsheets (with the
graphs on the spreadsheet for each) to your instructor by the due date given.
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BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS
EXCEL GRAPHING PRACTICE
Construct a data table and graph to determine the unknown DNA or Protein bands in the following
three examples. Formulas can be viewed by going to TOOLS, OPTIONS, VIEW and clicking
FORMULAS.
1. Use the results below to find the sizes of DNA bands that migrated 3.25, 3.55, and
4.55 cm on the gel. (NOTE: For DNA data results, the Rf is not usually used, but
rather the distance the fragments actually migrated in the gel.)
Mwt band Distance Log bp
size (bp) migrated size
(cm)
9,416
6,557
4,361
3,000
2,322
2,027
725
570
2.39
2.7
3.12
3.55
3.8
3.99
5.3
5.5
3.97
3.82
3.64
3.48
3.36
3.31
2.88
2.76
2. Determine the size of two unknown DNA bands that migrated 2.45 and 2.03 cm on
this gel.
Mwt band Distance Log bp
size (kb) migrated size
(cm)
10
8
6
5
4
3
2.5
2
1.5
1
0.5
1.4
1.52
1.7
1.84
2
2.2
2.4
2.55
2.77
3.1
3.6
4
3.9
3.78
3.7
3.6
3.48
3.4
3.3
3.18
3
2.7
3. Use the following results to determine the size of DNA bands that migrated 4.65,
4.49, 4.75, and 4.82 cm each.
Distance of
migration
(cm)
Log of bp
size
Size of
fragment
(bp)
3.15
3
1000
3.6
2.9
800
4.2
5.03
2.78
2.6
600
400
6.35
2.3
200
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BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS
ANSWERS
1. Data Table for Practice Question 1
Unknown bp size
B
(antilog
Y)
solve for Y x:
distance
migrated
4,038 3.606225
3.25
Slope of the line (m):
Y intercept (b):
3,118 3.493833
1,316 3.119193
3.55
4.55
Y = mx+b
-0.37464
4.823805
Unknown C
2. Data Table for Practice Question 2
-0.57541Slope (m)
4.770114y intercept (b)
bp size
y=mx+b
y value
2,293
Distance supercoiled plasmid migrated (cm)
3.36036
2.45
Distance relaxed plasmid migrated (cm)
3,999 3.602032
2.03
3. Data Table for Practice Question 3
migration Log of bp
size
(cm)
bp size Y = mx+b
4.65 2.675908
474
4.49
2.71078
514 slope (m) = -0.21795
4.75 2.654113
4.82 2.638856
451 intercept = 3.689375
435
Sources:
Ausubel, F.M. et al. Current Protocols in Molecular Biology, New York. John Wiley & Sons, 1994-2001.
http://csm.jmu.edu/biology/courses/bio480_580/mblab/rubiscointro.htm
http://www.ingentaconnect.com/content/cabi/ivp/2004/00000040/00000002/art00004
http://sunflower.bio.indiana.edu/~rhangart/courses/b373/lecturenotes/cellwall/cellwall.html
http://www.findarticles.com/p/articles/mi_m3741/is_1_53/ai_n8699055
Sambrook, J., Russell, D.W. Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor,
N.Y. Cold Spring Harbor Laboratory Press, 2001.
Thiel, T., Bissen, S., Lyons, E. Biotechnology: DNA to Protein; A Laboratory Project. McGraw Hill.
2002.
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