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

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.

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

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.

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

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

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

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

Gel Electrophoresis Experiment Edible Colors

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

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

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.

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

Alternative Experiments Kool-Aid 1 2 3 4 5 6 1) Strawberry 2) Orange 3) Tropical Punch 4) Grape 5) Ice Blue Raspberry Lemonade