세포생물학 및 실험 2 2018-2학기 생명과학과 박태식 교수님 화요일 (1-4 교시) Cell lysis

Method of cell disruption 1. Physical : very slow ex) freeze-thaw 2. Chemical lysis : can denaturate protein structure ex) alkali, organic solvents, detergents 3. Enzymatic lysis : often not reproducible, enzyme stability, long incubation time, necessity of removing the lysis enzymes, expensive scale-up, often combination with other method necessary ex) lysozyme, gulacanases, chitinase 4. Mechanical : expensive equipment ex ) Sonication, homogenization, ball/bead milling, French press

Lysis buffer for protein extraction < Components of the lysis buffer > Tris-HCl (pH 7.6) : most proteins are sensitive to pH NaCl : establish an ionic strength in the buffer solution and improve the stability of protein Detergent : disrupt lipid bilayers and aid in solubilizing hydrophobic proteins ▷ organic amphipathic (with hydrophobic tail and a hydrophilic head) surfactants. ▷ be categorized as nonionic, ionic based on their hydrophilic head group feature. ▶Nonionic detergents are weakly denaturing (will not disrupt protein functions). ex) Triton X-100 and NP-40 ▶ Ionic detergents and cationic detergents are denaturing (will disrupt protein functions). ex) deoxiycholate (moderately denaturing), sodium dodecyl sulfate (strongly denaturing) Protease inhibitor : inactivate protease (inactivate divalent ion to activate enzyme) and prevent protein degradation. <micelle>

Protocol Plate 2 × 106 HepG2 cells/dish on 60 mm2 dish and incubate overnight (37℃, 5% CO2). Treat WY14643 on dose-dependent (0, 10, 20, 40 μΜ) for 24 hours. ▷ 1 & 2조 : 10 μΜ, 3 & 4조 : 20 μΜ, 5 & 6조 : 40 μΜ Lysis cell to extract protein Carefully remove culture medium on ice from adherent cells. Wash cells with 1 ml of PBS gently and remove PBS. Add the 250 μl of lysis buffer. Gather the lysate to one side using a cell scraper, collect the lysate and transfer to a microcentrifuge tube (E-tube). (label on a new tube) Vortex 15-30 second and keep on ice for 10 minutes. (Repeat 3 times, total 30 minutes) Centrifuge samples at 13000 rpm for 20 minutes at 4 ℃ to pellet the cell debris. Transfer supernatant to a new tube. (label on a new tube) Store the lysate at -20 ℃.