MCDB428Lect02 - MCDB 428 Lecture 2: Monday January 7, 2008...

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Lecture 2: Monday January 7, 2008 Today’s Outline Immuno-EM and Immunofluorescence Production of Monoclonal Antibodies Biochemical Techniques Cell Fractionation Proteomics Pulse-chase analysis Genetic Techniques Types of mutants, segregation Complementation analysis Synthetic lethality Genomics MCDB 428
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With So Many Different Compartments: How To Know Which is Which? ? ? ? ? ? ? ? ?
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Antibodies Can Be Used To Detect Specific Proteins Different types of tags can be coupled to secondary antibodies: Gold beads, for Immuno-electron microscopy Fluorescent molecules, for Immunofluorescence Enzymes for immunoblotting or ELISA
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Immunodetection of Subcellular Compartments 1 o antibody = anti-catalase 2 o antibody = gold-linked anti-rabbit Endosomes Peroxisomes Green = GFP-Rab5 Red = 1 o Ab, anti-EEA1 2 o Ab, rhodamine-linked anti-rabbit
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(selective medium)
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Differential Centrifugation: One Way of Separating Subcellular Organelles Organelles are separated Based on their relative Size/Density
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Rate-zonal Centrifugation Rate-zonal Centrifugation separates Particles based on their relative size (Mr) M(r) = Molecular radius, a combination of Both size and density -e.g. both a golf ball and ping-pong ball are about the same size, but the M(r) of the golf ball is much higher because it is much denser Differential centrifugation is a form of Rate-zonal centrifugation Sucrose, or other high density liquid is used to increase the amount of time it takes for particles to sediment to the bottom of the tube, allowing for greater resolution of particles of different sizes
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Equilibrium Density Centrifugation Organelles of different Densities are separated based on their
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MCDB428Lect02 - MCDB 428 Lecture 2: Monday January 7, 2008...

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