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

2. 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

3. 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>

4. 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 second and keep on ice for 10 minutes. (Repeat 3 times, total 30 minutes)
Centrifuge samples at 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 ℃.