Announcements Discuss mid term feedback FNT heads up methods 3Q got rid of one Day 7 quiz staggered arrivals 1 1 5hrs Office hours Pre lab Lecture SDS PAGE Affinity purification recap Today in Lab Mod 2 Day 6 SDS PAGE preparation Acrylamide toxic You will make whole cell extracts with equal cell s Based on OD600 reading normalize 1 OD 1 0 2 OD 0 5 Vmax 15mL 1 7 5mL 7 5mL H20 2 15mL in hood Gel separates proteins based on size Sample preparation SDS coat proteins with negative charge Me breaks S S bonds Boiling denature higher order structures Sample Buffer has SDS Me plus glycerol BPB dye SDS PAGE visualization analysis Visualization Coomassie stain binds certain AA Two ladders visualization quantification respond to the stain in real time pre stained 150 ng 100 KDa 750 ng 50 KDa 150 ng 20 KDa Kaleidoscope Kaleidoscope Unstained Affinity purification Basis His tag in vector 6x binds to metals Courtesy of Life Technologies Carlsbad CA Used with permission high imidazole coat agarose beads with Ni2 add protein mixture wash away non His proteins elute His tagged protein Today in Lab Lyse cell pellets in BPER BSA carrier protease inhibitors Add 4 mL lysis enzymes Run a 25 L aliquot through SDS PAGE Two ladders also boil these too Stick with equal volumes if you have 25 Purify IPC protein from the rest long Immediately take 10 L aliquot and measure concentration The rest is stabilized w BSA to be titrated against calcium next time MIT OpenCourseWare http ocw mit edu 20 109 Laboratory Fundamentals in Biological Engineering Spring 2010 For information about citing these materials or our Terms of Use visit http ocw mit edu terms
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