ANAT 262 Lecture Notes - Column Chromatography, Cytosol, Electrophoresis

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In vitro cell-free system: used to study a particular aspect of a process which is re- duced to its minimal components (bottom up way to look at the cell) Ideally, looking at only one process and you know every ingredient that goes in but usually you are purifying a whole organelle starting with cytosol and trying to reduce it to a few components that you do understand. Use an assay to determine whether and how efficiently the desired process took place. Purify er membranes - get mix of rough and smooth microsomes (only interested in rough ribosomes = protein synthesis) Suspend, load onto gradient, and then rough microsomes go to the bottom. Use em to see what the fractions werenext added proteases which will chop up anything produced outside the lumen (proteins become protease resistant if they get into the lumen) Add mrna that codes for antibody light channels and use in vitro systems to eliminates some of the components.

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