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Electrophoresis

Gel

Agarose

Sabrina SchmidtkePartnership for Environmental Education and Rural Health

Protein Chemistry LaboratoryTexas A&M University


Electrophoresis is a laboratory technique for separating mixtures

of charged molecules.

What is Electrophoresis?

• 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.


Charged molecules are separated based on their electrical charge and size.

Separation of a Mixture of Charged Molecules

Charge Separation

Size Separation

Analyze

Identify

PurifyMixture of Charged Molecules

Positive Molecules

Negative Molecules


Real Life Examples of Uses for Electrophoresis

• Law Enforcement Agencies

• Hospitals

• Genetics Research


Components of Electrophoresis

• 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.


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.

- +

-+

-

+-

+-

+

- +

- - +

- -

--

Negatively Charged Protein

-+

-+

+

Positively Charged Peptide

+

N

N

O+

Positively Charged

Amino Acid


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

Proteins Entering Porous Material

Smallest Move Fastest

How Separation Occurs


• 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.

• 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

Gels can be made from substances such as agarose or polyacrylamide.

Red Sea Weed

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.

Overview of Gel Electrophoresis


Illustration of Gel Electrophoresis

- - Negative Electrode - -

+ + Positive Electrode + +

- - Negative Electrode - -

+ + Positive Electrode + +

Before Electrophoresis After Electrophoresis

Wells


Gel Electrophoresis Experiment

Edible Colors


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.


Materials List

Chamber and Power Supply– 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


Materials ListSamples and Gel Preparation– Small test tubes/vials– 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


Safety Precautions

• Chemicals – Aquarium Sealant, Agarose, and 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.


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.


• 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.




• 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.


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


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.


Preparing the Gel

• Lay the glass slide on the table. • Use the binder clips to hold the

comb just above the slide.


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.


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.


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.


Observing the Gel

• 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

Yel

low

Red

Gre

en

Blu

e

All


• 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

Yel

low

Red

Gre

en

Blu

e

Mix

ed

Observing the Gel


• 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.

Mix

ed

Blu

e

Gre

en

Yel

low

Red

Blu

e

Yel

low

Red

Observing the Gel


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.


Skittles

1 2 3 4 5

1) Grape2) Lime3) Lemon4) Orange5) Strawberry



Kool-Aid

1 2 3 4 5 6 1) Strawberry2) Orange3) Tropical Punch4) Grape5) Ice Blue Raspberry Lemonade


peer.tamu.edu cerh.tamu.edu pcl.tamu.edu electrophoresis gel agarose sabrina schmidtke partnership...

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