4.8. Preparation of Cytosolic and Nuclear Extracts
Cells were treated and harvested as described above, lysed with hypotonic lysis buffer [25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES; pH 7.5), 5 mM EDTA, 5 mM MgCl2, and 5 mM dithiothreitol (DTT)], and incubated for 15 min on ice. NP-40 (2.5%) was added and the cells were lysed for an additional 10 min. Nuclei were collected by centrifugation at 7500× g for 15 min at 4 ◦C. The supernatant was collected as the cytosolic fraction. Nuclear proteins were resuspended extraction buffer (10 mM HEPES, pH 7.9, 100 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, and 0.2 mM DTT) and incubated for 20 min at 4 ◦C. Extracts were centrifuged at 16,000× g for 10 min, and protein levels were determined using a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. Both fractions were then used for Western blot analysis.
이처럼 세포질 내의 단백질을 추출하고, 핵 내의 단백질을 따로 따로 추출할 때,
이 때 사용되는 lysis buffer에 의해 핵막은 붕괴가 안 되는걸까요?
어떻게 선택적으로 같은 '인지질'에 영향을 주는 buffer 임에도 불구하고 핵막에 손상을 주지 않고 핵 내의 단백질을 얻을 수 있는지 원리가 궁금합니다!
좀 쉽게 상세히 설명해주시면 감사하겠습니다! 부탁드립니다!!