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Transcript
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. peer.tamu.edu cerh.tamu.edu pcl.tamu.edu 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 peer.tamu.edu cerh.tamu.edu pcl.tamu.edu Real Life Examples of Uses for Electrophoresis • Law Enforcement Agencies • Hospitals • Genetics Research peer.tamu.edu cerh.tamu.edu pcl.tamu.edu 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. peer.tamu.edu cerh.tamu.edu pcl.tamu.edu 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 - + + N Positively Charged Amino Acid peer.tamu.edu - + + Positively Charged Peptide cerh.tamu.edu + - + + - + - + + - + Negatively Charged Protein pcl.tamu.edu 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 peer.tamu.edu Proteins Entering Porous Material cerh.tamu.edu Smallest Move Fastest pcl.tamu.edu 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 peer.tamu.edu cerh.tamu.edu pcl.tamu.edu Illustration of Gel Electrophoresis - - Negative Electrode - - - - Negative Electrode - - Wells + + Positive Electrode + + + + Positive Electrode + + Before Electrophoresis After Electrophoresis peer.tamu.edu cerh.tamu.edu pcl.tamu.edu Gel Electrophoresis Experiment Edible Colors peer.tamu.edu cerh.tamu.edu pcl.tamu.edu Observing the Gel peer.tamu.edu cerh.tamu.edu All Blue Green Red Yellow Possible results. pcl.tamu.edu Observing the Gel peer.tamu.edu cerh.tamu.edu 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. pcl.tamu.edu 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. peer.tamu.edu cerh.tamu.edu pcl.tamu.edu Alternative Experiments Skittles 1) 2) 3) 4) 5) peer.tamu.edu Grape Lime Lemon Orange Strawberry 1 cerh.tamu.edu 2 3 4 5 pcl.tamu.edu Alternative Experiments Kool-Aid 1) 2) 3) 4) 5) peer.tamu.edu Strawberry Orange Tropical Punch Grape Ice Blue Raspberry Lemonade cerh.tamu.edu 1 2 3 4 5 6 pcl.tamu.edu