Electrophoresis
Agarose
Gel
Sabrina Schmidtke
Partnership for Environmental Education and Rural Health
Protein Chemistry Laboratory
Texas A&M University
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What is Electrophoresis?
Electrophoresis is a laboratory
technique for separating mixtures
of charged molecules.
• Mixture: a material composed of
two or more elements or parts.
• Charged Molecules: a molecule (such as
a protein or DNA) that has too many
or too few electrons.
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Separation of a Mixture of
Charged Molecules
Charged molecules are separated based on
their electrical charge and size.
Positive Molecules
Analyze
Charge
Separation
Size
Separation
Mixture of
Charged Molecules
Identify
Purify
Negative Molecules
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Real Life Examples of Uses for
Electrophoresis
• Law Enforcement Agencies
• Hospitals
• Genetics Research
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Components of Electrophoresis
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Electrical Current – the flow of electric charge
Positive Electrode – the wire that collects electrons
Negative Electrode – the wire that emits electrons
Porous – containing pores, permeable to fluids
and small particles
• Sieve – a mesh device to filter small particles out of a
mixture of larger particles.
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How Separation Occurs
Electrical Charge:
Many molecules (amino acids, peptides, proteins, DNA, and
RNA) have naturally occurring negative and positive charges on
them. The sum of these charges determines the overall charge.
When introduced to an electrical current, negatively charged
molecules are attracted to the positive electrode and positively
charged molecules are attracted to the negative electrode.
N
+
O
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+
+
N
Positively Charged
Amino Acid
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-
+
+
Positively Charged Peptide
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+ - +
+ - + - + +
- +
Negatively Charged Protein
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How Separation Occurs
Molecule Size:
The porous material is made of microscopic particles suspended
in a gel. The microscopic particles attach to one another
forming tunnels that act as a sieve to separate the molecules.
Small molecules can move faster than large molecules.
Porous
Material
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Proteins Entering
Porous Material
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Smallest Move
Fastest
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Gel Electrophoresis
Gels can be made from substances such as agarose or
polyacrylamide.
• Agarose – a complex sugar chain from red
seaweed. It is commonly used in foods (ice cream,
whipped cream, and jellies) and many biological
mediums. It has a large pore size good for separating
large molecules quickly.
Red Sea Weed
• Polyacrylamide – chain of acrylic acid
molecules. It is often used to make plastics and
rubber. It has a small pore size good for separating
small molecules slowly. *Polyacrylamide is a
neurotoxin!
Acrylic Acid
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Overview of Gel Electrophoresis
• Mix agarose or polyacrylamide powder with liquid buffer.
• Pour the gel into a mold.
• Place a comb in the gel to form sample wells.
• Allow the gel to solidify.
• Submerge the gel in a tank full of a liquid buffer.
• Place the samples in the wells.
• Turn on the power source.
• Charged molecules will move to the oppositely charged electrode.
• Turn off the power source and remove the gel.
• Observe the separated molecules.
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Illustration of Gel Electrophoresis
- - Negative Electrode - -
- - Negative Electrode - -
Wells
+ + Positive Electrode + +
+ + Positive Electrode + +
Before Electrophoresis
After Electrophoresis
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Gel Electrophoresis Experiment
Edible Colors
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Overview of the Experiment
Purpose:
To introduce the principles and terminology of
electrophoresis and demonstrate the separation of
food coloring dyes with agarose gel electrophoresis.
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Materials List
Chamber and Power Supply
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Plastic dish
Slide box
Aquarium sealant
Large needle
Seizing wire
Hot glue gun and glue sticks
Nine-volt batteries
Nine-volt battery clip
Alligator clips
Scissors
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Materials List
Samples and Gel Preparation
– Small test tubes/vials
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Tube rack
Permanent marker
Transfer pipettes
Capillary tubes with bulb
Food colors
50% glycerol solution
2” x 3” glass slide
Well comb
Tris-Borate-EDTA Buffer (TBE)
Agarose
Graduated cylinder
Erlenmeyer flask
Balance
Microwave or hot water bath
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Safety Precautions
• Chemicals – Aquarium Sealant, Agarose, and
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•
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TBE Buffer are all irritants. If you get them on
your skin or in your eyes, rinse with water.
Electricity – do not touch the alligator clips or the
buffer when the power supply is assembled and
hooked up. IT WILL ELECTROCUTE YOU!!!!
Hot Objects – the hot glue gun and the agarose
solution will both be hot. If you get burned, rinse
the burn with cool water and seek medical attention
if necessary.
Glassware – (beakers, graduated cylinders, slides)
If anything is broken dispose of the glassware in an
appropriate manner.
Sharps – the needle, seizing wire ends, and
capillaries are all sharp. Be careful when handling
these items and dispose of them properly.
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Building the Electrophoresis
Chamber
• Place the slide box in the
center of the plastic dish and
trace around the edge with
the permanent marker.
• Extend the two long sides up
one side of the plastic dish.
• With the marker, place a spot
near the rim of the plastic
dish, about 1 cm out from
each of the lines.
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Building the Electrophoresis
Chamber
• Carefully use the large needle to
punch a hole at each spot.
• Cut a piece of wire about 10 cm
longer than the length of the
plastic dish.
• Bend the wire as shown.
• The wire should touch the plastic
dish on both sides, but be about 1
cm off of the bottom.
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Building the Electrophoresis
Chamber
• Put a thick bead of aquarium
sealant around the rim of the
slide box.
• Place the slide box into the
plastic dish.
• Allow the aquarium sealant
to dry overnight.
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Building the Power Supply
• Cut the battery clip in half with
the scissors. Be careful not to
cut the wires.
• Remove one cover off each
alligator clip.
• Feed the battery clip wire
through the cover and wrap it
around the appropriate alligator
clip.
• Replace the cover to protect the
wire connection.
• Connect the batteries into a
pyramid.
• Connect the battery clips to the
batteries
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Preparing the Gel
• Place agarose and TBE
buffer into the Erlenmeyer
flask.
• Heat the flask in the
microwave or a hot water
bath. The flask will become
very hot.
• When the agarose is
dissolved, there will be no
more particles in the TBE
Buffer.
• Allow the flask to cool until
you are able to touch it
without burning yourself.
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Preparing the Gel
• Lay the glass slide on the table.
• Use the binder clips to hold the
comb just above the slide.
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Pouring the Gel
• Use a transfer pipette to place the agarose onto the slide.
• Start at the comb and work your way out covering the whole
slide. Pop any air bubbles immediately.
• When the gel has solidified it will become opaque.
• Carefully remove the comb.
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Preparing and Loading the
Samples
• Label the test tubes with the
color of food dye you will put
in them.
• Mix one drop of food coloring
with three drops of 50%
glycerol. Repeat with all
samples.
• Carefully place the gel in the
plastic dish on top of the slide
box.
• Fill the chamber with TBE
Buffer until the gel is covered.
• Use the capillary tubes to load
10 μl of sample in each well.
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Developing the Gel
• Assemble the battery pyramid
and connect alligator clips.
• Connect the black alligator
clip to the wire behind the
wells and the red alligator clip
to the wire in front of the
wells.
• DO NOT touch the buffer
while the power supply is
attached to the chamber!
• Allow the gel to develop for at
least 35 minutes. You will be
able to see the dyes separate.
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All
Blue
Green
Red
• Disconnect the alligator clips and
take apart the battery pyramid.
• Carefully remove the slide with
gel and lay on a paper towel.
• Observe the dye separation of
the individual food colors.
• Compare the dye separation of
the mixtures with the dye
separation of the individuals
Yellow
Observing the Gel
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Mixed
Blue
Green
Red
• Yellow food coloring
separated into yellow dye
• Red food coloring separated
into red dye and pink dye
• Green food coloring
separated into yellow dye
and blue dye
• Blue food coloring separated
into red dye and blue dye
• The mixed sample contains
all of the dyes
Yellow
Observing the Gel
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Observing the Gel
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Red
Yellow
Green
Blue
Mixed
Red
Yellow
Blue
• This gel was run for 120
minutes, it shows better
separation of the dyes and
good replication for the dyes.
• The size of molecules from
smallest to largest are:
yellow, red, pink, and blue.
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Alternative Experiments
• Other Samples
– Separate the food dyes used in Kool-Aid and Skittles.
– Separate proteins and DNA. (will require additional materials)
• pH Change
– Change the pH of the buffer in the gel and the tank to observe
the changes it makes on the samples.
• Change the Percentage of Agarose Used
– Observe how using higher/lower concentrations of agarose will
change the separation of dyes.
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Alternative Experiments
Skittles
1)
2)
3)
4)
5)
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Grape
Lime
Lemon
Orange
Strawberry
1
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2
3
4
5
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Alternative Experiments
Kool-Aid
1)
2)
3)
4)
5)
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Strawberry
Orange
Tropical Punch
Grape
Ice Blue Raspberry Lemonade
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1
2
3
4
5
6
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