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Electrophoresis

Gel

Agarose

Sabrina SchmidtkePartnership for Environmental Education and Rural Health

Protein Chemistry LaboratoryTexas A&M University

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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.

CSI Video Link

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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

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Real Life Examples of Uses for Electrophoresis

• Law Enforcement Agencies

• Hospitals

• Genetics Research

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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.

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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.

- +

-+

-

+-

+-

+

- +

- - +

- -

--

Negatively Charged Protein

-+

-+

+

Positively Charged Peptide

+

N

N

O+

Positively Charged

Amino Acid

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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

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• 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

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Illustration of Gel Electrophoresis

- - Negative Electrode - -

+ + Positive Electrode + +

- - Negative Electrode - -

+ + Positive Electrode + +

Before Electrophoresis After Electrophoresis

Wells

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Gel Electrophoresis Experiment

Edible Colors

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Observing the Gel

Possible results.

Yel

low

Red

Gre

en

Blu

e

All

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• 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

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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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Skittles

1 2 3 4 5

1) Grape2) Lime3) Lemon4) Orange5) Strawberry

Alternative Experiments

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Kool-Aid

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

Alternative Experiments