1. Electrophoresis Agarose Gel
Sabrina Schmidtke
Partnership for Environmental Education and Rural Health
Protein Chemistry Laboratory
Texas A&M University
peer.tamu.edu cerh.tamu.edu pcl.tamu.edu

2. What is Electrophoresis?
Electrophoresis is a laboratory technique for separating mixtures of charged molecules.
CSI Video Link
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.

3. Separation of a Mixture of Charged Molecules
Charged molecules are separated based on their electrical charge and size.
Charge Separation
Size Separation
Analyze
Identify
Purify
Mixture of Charged Molecules
Positive Molecules
Negative Molecules

4. Real Life Examples of Uses for Electrophoresis
Law Enforcement Agencies
Hospitals
Genetics Research

5. 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.

6. Positively Charged Amino Acid
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 O
Positively Charged Amino Acid

7. Proteins Entering Porous Material
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
Proteins Entering Porous Material
Smallest Move Fastest

8. 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.
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!
Red Sea Weed
Acrylic Acid

9. Illustration of Gel Electrophoresis
- - Negative Electrode - -
- - Negative Electrode - -
Wells
+ + Positive Electrode + +
+ + Positive Electrode + +
Before Electrophoresis
After Electrophoresis

10. Gel Electrophoresis Experiment
Edible Colors

11. Observing the Gel Possible results. Red All Green Blue Yellow
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Red
All
Green
Blue
Yellow

12. 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.
Blue
Red
Blue
Green
Red
Yellow
Mixed
Yellow

13. 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.

14. Alternative Experiments
Skittles
1) Grape
2) Lime
3) Lemon
4) Orange
5) Strawberry

15. Alternative Experiments
Kool-Aid
1) Strawberry
2) Orange
3) Tropical Punch
4) Grape
5) Ice Blue Raspberry Lemonade