PP08 - 10/07/10 1 Electrophoresis and Electroblotting of...

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Unformatted text preview: 10/07/10 1 Electrophoresis and Electroblotting of Proteins QuickTime and a decompressor are needed to see this picture. http://www.vias.org/science_cartoons/electrophoresis.html 10/07/10 2 Things to Learn from This Weeks Lab: 1. How to prepare and load an acrylamide gel. 2. The principles of gel electrophoresis 3. The use of size standards to estimate protein size. 5. Electroblotting of proteins from a gel to a membrane 4. How to stain proteins in a gel. 10/07/10 3 What is electrophoresis? The movement of molecules through a medium , in response to an electric field. current 10/07/10 4 Gel Electrophoresis: Molecules are Electrophoresed through a Gel The gel is approximately the consistency of jello . The gel can be made of different materials and to different densities , depending on what is to be separated. 10/07/10 5 Types of Gels Agarose: forms a loose , porous gel the gel is placed horizontally suitable for separating larger molecules like DNA and most RNAs 10/07/10 6 Types of Gels Polyacrylamide Forms a tighter , denser gel The gel is placed vertically Suitable for separating smaller molecules, like proteins and small DNAs or RNAs . 10/07/10 7 Acrylamide Polymerizes to Form a Gel Acrylamide monomer A polyacrylamide/Bis-acrylamide gel + bis-acrylamide with TEMED Acrylamide dimer Spontaneously in solution 10/07/10 8 ANODE CATHODE POWER SUPPLY LOWER BUFFER RESERVOIR (+) UPPER BUFFER RESERVOIR (-) LANE: The region in a gel where the molecules from one well travel. Wells: Sample Loading Sites 10/07/10 9 POWER SUPPLY LOWER BUFFER RESERVOIR ANODE CATHODE (+) UPPER BUFFER RESERVOIR (-) LANE: The region in a gel where the molecules from one well travel. 10/07/10 10 Review Cathode (negative terminal) attracts cations (+) Anode (positive terminal) attracts anions (-) 10/07/10 11 Which molecule is present in the protein...
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PP08 - 10/07/10 1 Electrophoresis and Electroblotting of...

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