SID 7969543 (Standard)

The high-salt/hypotonic fractionation method for nuclear protein extraction is based on the differential solubility of cellular components. Cytoplasmic proteins are extracted first using a hypotonic buffer that causes cell swelling and membrane rupture, followed by centrifugation to separate the cytoplasmic supernatant from the nuclear pellet. The nuclear pellet is then subjected to high-salt extraction (e. g. , 0. 4 M (NH4)2SO4 or 1 M NaCl) to solubilize tightly bound nuclear matrix proteins, including transcription factors, histones, and structural proteins associated with chromatin and the nuclear scaffold. This approach allows for the isolation of both soluble cytoplasmic proteins and salt-resistant nuclear proteins while minimizing cross-contamination.

By lysing cells, releasing RNA, and removing impurities such as proteins and DNA, high-purity RNA products are finally obtained. The commonly used traditional method is the guanidine isothiocyanate/phenol/chloroform method (Trizol), which is suitable for a variety of animal materials including animal tissues, microorganisms, cultured cells, etc., and most plant materials.

Cytotoxicity assays are usually based on the assessment of cell membrane damage, which can also be indirectly detected by measuring cell viability. Detection methods include MTT assay, CKK-8 assay, LDH assay and ATP assay, etc.