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 Vmax = 15mL (1)7.5mL + 7.5mL H20 (2)15mL (1) OD = 1.0 (2) OD = 0.5 • Gel separates proteins based on size, • Sample preparation – SDS: coat proteins with negative charge – β‐Me: breaks S‐S bonds – Boiling: denature higher‐order structures – Sample Buffer has SDS, β‐Me, plus glycerol, BPB dye in hoodKaleidoscopeSDS‐PAGE visualization, analysis • Visualization: Coomassie stain (binds certain AA) • Two ladders: visualization, quantification Kaleidoscope Unstained 750 ng (@50 KDa) 150 ng (@20 KDa) 150 ng (@100 KDa) in real‐time, pre‐stained respond to the stainAffinity purification • Basis: His‐tag in vector 6x, binds to metals high [imidazole] coat agarose elute His-tagged beads with Ni2+ add protein wash away mixture non-His proteins protein Courtesy of Life Technologies, Carlsbad, CA. Used with permission.Today in Lab • Lyse cell pellets in BPER – BSA “carrier,” protease inhibitors – Add 4 mL lysis enzymes • Run a 25 µL aliquot through SDS‐PAGE – Two ladders also Æ boil these too – Stick with equal volumes if you have < 25 • Purify IPC protein from the rest (long!) – Immediately take 10 µL aliquot and measure concentration – The rest is stabilized w/BSA, to be titrated against calcium next timeMIT OpenCourseWarehttp://ocw.mit.edu 20.109 Laboratory Fundamentals in Biological Engineering Spring 2010 For information about citing these materials or our Terms of Use, visit:
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