Membrane protein extract homogenization

The pellet was washed twice with 50 mM Ttis-HCl buffer, pH 8.0, containing 10 mM NaCl, to purify the membranes from stroma proteins. The thylakoid membranes were then homogenized with a small amount of washing buffer and sonicated in a cold ulttasonic bath for 30 min. For fiimre solubilization of the membrane proteins an equal volume of cold n-butanol (-20°Q was used. The phases were separated by centrifugation for 5 min at 1000 g and vratet phase contained the proteins was collected. The butanol extraction was repeated twice to obtain a lipid-free protein preparation. 

Mouse livers were excised and homogenized with a Potter-Elvehjem homogenizer. The microsomal fraction was isolated by centrifuging the homogenate at 9000 x g for 15 min, to sediment nuclei, mitochondria, and membrane debris. The supernatant was centrifuged at 100,000 x g for 60 min to pellet microsomes. The microsomes were further purified by another centrifugation step to remove soluble proteins (e.g., hemoglobin). HCQ was incubated with the microsomes for 2 h. Then a liquid-liquid extraction technique was performed to recover HCQ and its metabolites. The molecules were separated by a commercial CE system with ultraviolet (UV) absorbance detection. 

The level of LPO was measured with a fluorescence method [8]. Lipids were extracted by the mixture of chloroform and methanol (2 1). Lipids of mitochondrial membranes (3-5 mg of protein) were extracted in the glass homogenizer for 1 min at 10°C. Thereafter, equal volume of... 

---