Extensions of Gel Electrophoresis with Proteins
Brian McClain
Amos P. Godby High School
Tallahassee, Florida
Summer 2005
Research Host:
Dr. Timothy S. Moerland
Florida State University
Lesson # 8
Appropriate citation:
McClain, Brian Extensions of Gel Electrophoresis with Proteins (APS Archive of
Teaching Resources Item #7305. [Online]. Bethesda, MD: American Physiological
Society, 2005.
http://www.apsarchive.org/resource.cfm?submissionID=7305
Editor’s notes:
Website URLs listed in this resource were current as of publication, but may now be
obsolete. If you know of a replacement URL, please suggest it in the resource’s
“Comments” section.
Safety Concern: Teacher should be sure to go over the safety precautions listed in
the activity with their students.
Disclaimer:
This activity was created by the author and reviewed by the American Physiological
Society. Any interpretations, statements, or conclusions in this publication are those
of the author and do not necessarily represent the views of either the American
Physiological Society or the funding agencies supporting the professional development
program in which the author participated.
Frontiers in Physiology
www.frontiersinphys.org
© The American Physiological Society
Permission is granted for workshop/classroom use with appropriate citation
Extensions of Gel
Electrophoresis with Proteins
Teacher Section
Purpose
The purpose of this activity is to provide students awareness that the types of proteins within
tissue samples can be investigated by gel electrophoresis. In this process, students will become
familiar with the procedure of agarose electrophoresis.
Objectives
The student will be able to:

Describe the overall process of gel electrophoresis.

Qualitatively compare the protein contents of a variety of samples.

Speculate as to why different tissues or organisms may have some proteins in common
while yet having some differences in protein content.
Grade Level
This activity was designed for high school Biology; second year Biology electives such as Anatomy
and Physiology, Advanced Placement Biology, and second-year Biology courses.
National Science Education Standards
K-12 Unifying Concepts and Processes:
Systems, order, and organization
Change, constancy, and measurement
Evolution and equilibrium
Form and function
Grades 9-12
Science as Inquiry
Abilities necessary to do scientific inquiry
Physical Science
Structure and properties of matter
Motions and forces
Interactions of energy and matter
Life Science
The cell
Biological evolution
Matter, energy & organization in living systems
Science and Technology
Abilities of technological design
Florida Science Education Standards
Grades 9-12
SC.A.2.4.2
Student knows the difference between an element, molecule, and a compound.
SC.C.2.4.5
Student knows that most observable forces can be traced to electric forces acting
between atoms or molecules.
SC.F.1.4.5
Student knows that complex interactions among the different kinds of molecules in
the cell cause distinct cycles of activity governed by proteins.
SC.G.1.4.1
Student knows of the great diversity and interdependence of living things.
Brian McClain
Extensions of Gel Electrophoresis with Proteins
©2006 The American Physiological Society.
2005 Frontiers in Physiology Research Teacher
APS Works in Progress
Permission is granted for duplication for workshop/classroom use.
Teacher Section
Prior Knowledge
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Principles of protein structure
Principles of electrophoresis
Lab skills needed for gel electrophoresis
Concepts of unity of life and diversity of life.
Time Required
Prior to attempting agarose gel electrophoresis with protein samples, an introductory lab
experience with agarose gel electrophoresis is necessary. Various educational suppliers carry
products that accomplish this purpose, usually described as “Introduction to Gel Electrophoresis.”
These experiences have students use a low-percentage (0.8-2%) agarose gel to separate individual
dyes and a mixture of some of the individual dyes. Students analyze the results, hopefully
learning that molecules have sizes and charges and thus move in electrical fields in different yet
predictable ways. Often a "paper lab" relating these principles to restriction fragment length
polymorphisms (RFLP) of DNA restriction enzyme digests, DNA fingerprinting, is made. This type
of lab is traditionally done early in a Biology course with the biochemistry or the DNA unit. This
experience takes 50-90 minutes (1-2 class periods) depending on equipment and amount of
prior preparation of the materials by the teacher.
New Experiences with Proteins: These subsequent experiences also may each take 50-90
minutes (1-2 class periods) depending on equipment and amount of teacher prior preparation of
the materials, however new or reinforcing content can be learned during the time that it takes
the gels to run (20-40 minutes).
The first "How to" experience of protein electrophoresis is suggested to illustrate that larger
molecules like proteins can also be separated by electrophoresis based on relative molecular size
and charge. Known standard mixtures of both pre-stained and unstained proteins are
commercially available and are the positive controls. Comparison of migration distance and Rf
values can be determined similarly to the introductory lab and compared to the known molecular
weights in your control sample.
The second experience with electrophoresis of proteins presented here is to address, in an
inquiry manner, that tissue from the same organism or from different organisms can be compared
qualitatively using electrophoresis. Students work in groups to investigate a question of their own
design (see Student Section). The teacher will need to collect and provide a range of known and
unknown materials for safety reasons (hygiene, sanitation, financial costs and disposal). With this
next experience students may then appreciate some of the unity and diversity of life (e.g., many
cells may share some types of proteins, some cells may share many types of proteins, and some
organisms will have similar proteins in similar tissues). This awareness of the "proteome" (as
compared to the "genome") may lead students to choose a topic of their own to investigate. For
teachers and classes who prefer a more guided inquiry at this point, some possible topics of
investigations may compare samples of proteins in items available at the grocery store such as:
different milks, eggs, muscles, or organs. Some education suppliers do have similar kits available
for purchase, but by learning the technique of gel electrophoresis by this sequence of
experiences, students are able to investigate questions of their own.
Brian McClain
Extensions of Gel Electrophoresis with Proteins
©2006 The American Physiological Society.
2005 Frontiers in Physiology Research Teacher
APS Works in Progress
Permission is granted for duplication for workshop/classroom use.
Teacher Section
Including All Students
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
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This lesson requires cooperative, hands-on involvement by all students in groups of two or
three students. The first process is driven by students' explorations of provided materials.
The students subsequently will work in follow-up investigations that are student-designed,
at least in terms of selecting an investigable question.
Teachers may want to group students with gender, cultural background, and relative
ability in mind. The follow-up investigations can be accomplished with different student
groupings.
During the lab procedures, the teacher should ensure that all students have the
opportunity to accomplish the various tasks and skills associated with gel electrophoresis.
By using dyes and prestained proteins, this procedure may appeal to visual and kinesthetic
learning styles. Inevitably, some of the lab procedures will conveyed orally and visually.
Questions to Ask Along the Way
K "Pre/Prior" to Questions
Will each cell in an organism contain the exact same types of proteins?
Why might different cells have different proteins?
Will relatively young cells have the same types of proteins as older cells?
Why might the age of a cell cause different proteins to be expressed?
Are all cells of a given organism capable of producing the same proteins?
Where is the information for making any particular protein?
W "In the Middle of It" Questions
How can we modify our procedure to improve our results?
How can we quantify our observations?
Do our data support our hypotheses?
L "After It's Over" Questions
What are the similarities among our samples?
What are the differences among our samples?
What might explain these similarities and differences?
What can be our next step or deeper question to pursue?
Materials
The materials that are needed for protein electrophoresis using agarose begin with the same set
of materials that are used for any introductory gel electrophoresis lab and/or the DNA
fingerprinting labs (for example, Ward's Introduction to Agarose Electrophoresis, Ward's Natural Science
Establishment, Inc., 1998). The three major points of variation for protein agarose gel
electrophoresis are that:
1) A higher concentration (3.5%) of agarose is used;
2) A slightly different type of agarose is used (recommend Carolina Biological agarose for protein
electrophoresis products); and
3) A different running buffer (Tris-glycine-SDS) is required.
Brian McClain
Extensions of Gel Electrophoresis with Proteins
©2006 The American Physiological Society.
2005 Frontiers in Physiology Research Teacher
APS Works in Progress
Permission is granted for duplication for workshop/classroom use.
Teacher Section
It should be noted that research labs conducting protein research use polyacrylamide gel
electrophoresis (PAGE). While pre-cast acrylamide gels are available for purchase, the safety
concerns of any acrylamide and of its disposal vary. Also, the apparatus or gel tray and gel box
are different for PAGE, an expense that many high schools are not able to meet. The procedure
presented here uses the same equipment that many schools already have and with which many
Biology teachers are familiar.
"How To" Experience:

Prestained protein standards

Prestained "unknown" proteins
Sigma Chemical and Bio-Rad Laboratory both offer products-- see their website or catalog. A local
university or community college may also have such samples, and since the volume you need is
relatively small depending on experimental design (10-50 µl) they may be able to provide these
control samples and/or suggest samples for the subsequent inquiry experience.
For Each Group:

Samples in labeled tubes: standards, samples; in sample buffer.

Agarose Gel (3.5%) on gel tray

Tris-glycine-SDS Running buffer at 1X (will need to dilute from stock), 350 ml

Laemmli sample buffer
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Micropipettes and tips
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Microwave oven
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Metric ruler
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Calculator
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Goggles
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Gloves
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Aprons
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Coomassie stain solution
Shared Among Groups:

Power supply
Note: some electrophoresis set-ups are capable of running two or more gels at a time.
Additionally, many gel trays are designed to take two well combs, thus allowing more than
one group to run samples in a gel.
Safety
Electrical Safety: The electrophoresis power supply is an electrical device. Dry hands, checking
for frayed cords, and other standard safety procedures must be followed.
Safety goggles: If samples are placed in a hot water bath or if reducing chemicals (i.e., betamercaptoethanol) are used, protective eye goggles must be worn.
Sanitary conditions: If samples of raw foods (as tissue sources) are used, disposable gloves and
substantial hand washing must happen. Raw chicken and many other food items have microbial
contaminants whose transference must be prevented.
Chemical safety: Teachers should read and become familiar with the procedures for proper use,
storage and disposal of all chemicals. Many supply companies now send the material safety data
sheets with each order. If a school facility does not have a specific MSDS, the supplier will fax
one upon request and many MSDS sheets are available online. Follow MSDS directions for storage
and disposal of chemicals.
Brian McClain
Extensions of Gel Electrophoresis with Proteins
©2006 The American Physiological Society.
2005 Frontiers in Physiology Research Teacher
APS Works in Progress
Permission is granted for duplication for workshop/classroom use.
Teacher Section
Preparation
Early on the teacher needs to decide to what degree he or she will set-up materials prior and
what steps the students will execute for themselves. The procedure of gel electrophoresis is
integral to modern biology; this author recommends that the students are orally guided through
the procedures. Just as a high school class uses microscopes in different units of study, this set of
suggested lessons encourages the use of gel electrophoresis at different points during the course.
Note: 3.5% agarose is much thicker than that usually used for an introductory dye separation or
DNA RFLP run. It is recommended to heat 30 ml of 1X running buffer to near boiling in a
microwave. Then add the agarose (1.05 g) in small increments, stir and smush, and reheat in a
microwave for ten seconds. Repeat until all the agarose is in solution. Bubbles will appear. On
pouring the gel, tap the gel box firmly on the table surface to bring bubbles to the surface.
While the agarose is still warm, one can try to drag the bubbles to one end. The bubbles will be
much less prevalent and noticeable after the gel has set in buffer for staining and/or destaining.
For this reason, you may chose to pour the gels the day before and leave overnight in running
buffer or pre-weigh the agarose and aliquot the buffer. A microwave oven in the classroom for
the day will greatly expedite this portion of the procedure.
Procedure
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Set the ends of the gel tray as specified by the manufacturer. This may involve end dams
or tightly sealed masking tape.
Place the gel comb(s) in the desired slots in the gel tray. Remember that your samples
will generally run from black to red poles.
Pour approximately 30 ml of melted agarose into the tray. The agarose should come onethird to halfway up the teeth of the comb.
The gel should solidify in 15-20 minutes, taking on an opaque appearance.
Remove dams or masking tape from the gel tray ends. Carefully lower into gel box. Note
the orientation; remember your samples will run black to red terminals.
Pour running buffer into the gel box, filling both ends and covering the gel. Gels can be
kept overnight in this manner and then be ready to run the next day.
Running a gel

Remove the well combs, pull firmly and straight up. Tap the gel tray to cause any
lingering air bubbles in the wells to rise to the surface.

Use a micropipette to load samples from the tubes into the wells. A volume of 10-20 µl is
a conventional range for a sample volume. Take care not to pierce the bottom of the
well.
Note: Many students need practice with the hand-eye coordination required for micropipette use.
Previous experience with the dye separation electrophoresis lab should provide the practice
needed. Some suppliers do sell practice kits or one can pour some low concentration and less
expensive gels or Petri dishes with the combs in place to provide opportunities for practicing
micropipette use.


Carefully wipe up any liquid on the counter or on the gel box lid. With dry hands, slide the
cover of the gel box onto the electrodes. Again, confirm the placement of your samples so
that they will run from the black pole to the red pole.
With dry hands, connect the cords from the gel box to the power supply.
Brian McClain
Extensions of Gel Electrophoresis with Proteins
©2006 The American Physiological Society.
2005 Frontiers in Physiology Research Teacher
APS Works in Progress
Permission is granted for duplication for workshop/classroom use.
Teacher Section
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With dry hands, plug the power supply into the primary outlet.
Set or confirm the voltage. Short runs usually are run somewhere between 70-125 V,
depending on the equipment capability.
Turn on the power supply. After 10-20 seconds, bubbles should rise up from the wire
electrodes in the two deep reservoirs of the gel box.
Observe the migration of the loading dye toward the red pole. Normal run times at this
voltage are from 20-40 minutes. A final end point may be when the loading dye has
reached the half-way or end point of the gel.
With dry hands, follow the previous steps but in reverse - turn off, wait 10-20 seconds,
unplug, carefully slide gel box top off.
One can notch one corner of the gel to declare a benchmark for orientation. After some
staining and destaining procedures, the original dyes and markings on the gel may be
much less noticeable.
If one has used prestained molecular weight markers and prestained proteins, one can
proceed to using a metric ruler to make measurements. If unstained standards or samples
were used, then staining and destaining processes now follow. A Coomassie stain that
contains methanol (45% by volume), glacial acetic acid (5%), deionized water (50%), and
Brilliant Blue is recommended. Various commercial products are available and different
recipes are available on the web.
"How To" Experience for Inquiry with Student Selected Samples

Sample preparation: When trying to determine the relative molecular weight of a
protein, the samples are usually denatured. This process involves heating in the presence
of a reducing agent. A normal glass beaker hot water bath can be used. The sample
buffer, or Laemmli sample buffer, generally contains some type of pH buffer brought to
50 mM β-mercaptoethanol while containing sucrose or glycerol for density. Samples can
be sufficiently prepared by 5-15 minutes in 90-100˚C water bath. Care should be taken to
use screw top tubes or firmly closing tubes that can withstand such heating. Protective
safety wear must be worn.

Commercially obtained standards and proteins can be obtained at appropriate and known
concentrations for gel runs. Consult the packaging directions and MSDS protocols.

Locally obtained samples for experimentation or inquiry may be prepared by
homogenizing 0.1-0.3 g of tissue in 0.5-1.0 ml of sample buffer. However, dilutions will
likely be needed to avoid overloaded and smeared appearance of final gels.
"Inquiry" Samples

Some possible comparisons of samples may include, but should not be limited to:
different tissues from the same organism, different developmental stages of the same
species, or cross-species comparisons of similar tissues.

As with most inquiry lessons, a goal is for the student(s) to develop investigable questions.
You may need to provide and example, such as: Do omega-3 lipid eggs have the same
types of proteins as normal eggs? This may mean a brainstorming session after completing
the "How To" activity.

For a more structured approach, one may take advantage of a chicken wing-dissection lab
later in the course as a source of different muscle and tissue types for comparisons
(skeletal muscle vs. ligament vs. tendon vs. etc.)
Brian McClain
Extensions of Gel Electrophoresis with Proteins
©2006 The American Physiological Society.
2005 Frontiers in Physiology Research Teacher
APS Works in Progress
Permission is granted for duplication for workshop/classroom use.
Teacher Section
Where to Go From Here
This approach and technique may be used at several points throughout the course when the
concepts of unity, diversity, and protein expression need to be emphasized and reviewed. For
example, a cross-species comparison of similar tissues can be accomplished. In fact several
commercially available products are available for this (BioRad, Carolina). In development, seeds of
plants or larvae of insects can be compared to intermediate and adult stages. In a body system
unit, the protein content in types and relative amounts from tissues from different organs may be
compared. In each of these instances, when common proteins are "found" as revealed by shared
bands in the gel - the unity of life is demonstrated. When different bands are "found," the
diversity of life can be seen. Individual student research for science competitions is highly
encouraged.
Suggestions for Assessment
In the initial activity, group-authored "scientific papers" may be submitted in a variety of
formats: hand-written, PowerPoint slide-outlined, or emailed directly to the teacher as an
attachment (paperless assignment). Scans of the produced gels are strongly encouraged. For
subsequent investigations, either a student/group-specific KWL can be used or a similar scientific
paper/lab report may be submitted. Students should see any rubric used for evaluations prior to
submitting their work. Excellent sample rubrics for lab reports can be found in other APS
resources (R. Gillis, Brain Week Lab Report Rubric, in 2005 Works in Progress, APS Frontiers in
Physiology, p. 81). The teacher is strongly encouraged to complete her or his own KWL, either
modeling the role of a scientist or as the educator in order to evaluate the effectiveness of this
lesson.
References and Resources

Campbell, N. and J. Reece. (2002). Biology (6th ed.). Benjamin Cummings. San Francisco.

Johnson, G. and P. Raven. (1998). Biology, Principles and Explorations. Holt, Rinehart and
Winston. Austin, TX.

The Protein Protocols Handbook, 2nd Edition. Edited by John M. Walker. Humana Press.
Totowa, NJ. 2002. "SDS Polyacrylamide Gel Electrophoresis of Proteins" by John M.
Walker. Pp. 61-67.

Genetic Science Learning Center at the University of Utah.
http://gslc.genetics.utah.edu/units/biotech/gel

Cold Spring Harbor Laboratory, DNA Learning Center. http://www.dnai.org/teacherguide

Bio-Rad Laboratory, Biotechnology Explorer Program. http://www.bio-rad.com

Sigma-Aldrich Chemical Company. http://www.sigmaaldrich.com

Carolina Biological Supply Company. http://www2.carolina.com
Brian McClain
Extensions of Gel Electrophoresis with Proteins
©2006 The American Physiological Society.
2005 Frontiers in Physiology Research Teacher
APS Works in Progress
Permission is granted for duplication for workshop/classroom use.
Student Section
Internet Treasure Hunt
Gel Electrophoresis
This web info search will help you find information about gel electrophoresis. You will be
looking at pre-selected web sites to answer each question. It is important to not only find the
information at the site, but also to consider who wrote the site, what their purpose is in writing
it, and how credible (accurate) you think the information is.
Site 1:
gslc.genetics.utah.edu/units/
biotech/gel/
Who created
this web site
(organization,
etc.)?
Why did they
create it? (check
all that apply)
How credible
(accurate) do
you think the
info is?
Site 2:
web.utk.edu/~khughes/GEL/
sld001.htm
To provide factual information
To influence the reader’s opinion
To sell a product or service
I’m not sure
To provide factual information
To influence the reader’s opinion
To sell a product or service
I’m not sure
Very accurate
Somewhat accurate
Not very accurate
I’m not sure
Very accurate
Somewhat accurate
Not very accurate
I’m not sure
What did you
learn?
Brian McClain
Extensions of Gel Electrophoresis with Proteins
©2006 The American Physiological Society.
2005 Frontiers in Physiology Research Teacher
APS Works in Progress
Permission is granted for duplication for workshop/classroom use.
Student Section
Student Lab Handout
Group Members:
Gel Electrophoresis of Proteins Experimental Design
Background: Earlier in the course we did the Introduction to Gel Electrophoresis lab
where we separated small dye-colored molecules. We used a gel made of agarose and an
electric current to separate the molecules based on size and electrical charge. Now that
we have learned about proteins, we will use these same principles to study proteins.
What are the samples that will be made available to you?
What is it that your group wants to test?
(Perhaps state your independent variable and dependent variable.)
What is your hypothesis?
(Try to make a prediction and offer a "why" for your prediction.)
List the materials you will need.
Brian McClain
Extensions of Gel Electrophoresis with Proteins
©2006 The American Physiological Society.
2005 Frontiers in Physiology Research Teacher
APS Works in Progress
Permission is granted for duplication for workshop/classroom use.
Student Section
Student Lab Handout
List your experiment's procedure. Make sure to include amounts and to number your
steps.
(For reference, use your previous lab "Introduction to Agarose Electrophoresis.")
List at least three safety rules that everyone in your group will practice.
Sketch out your data table. List the independent variable(s) and leave spaces for
your data.
Teacher's Initials for Approval of Plan Prior to Beginning the Experiment _______
Brian McClain
Extensions of Gel Electrophoresis with Proteins
©2006 The American Physiological Society.
2005 Frontiers in Physiology Research Teacher
APS Works in Progress
Permission is granted for duplication for workshop/classroom use.