A study published in BMCBiology discusses how mitochondrial genes in humans might have gone unnoticed and finds alternative mitochondrial peptides that may offer a new framework for the investigation of mitochondrial functions and diseases.
HeLa cells were washed with PBS and harvested in a lysing buffer containing 10 mM HEPES, 150 mM NaCl, and a cocktail of protease inhibitors . Cells were then lysed with a sonic dismembrator sonicator 3 times for 30 s before adding 100 µl of Triton X-100 10%. Cell lysates were incubated on ice for 20 min and then centrifuged for 15 min at 15,000 RPM at 4 °C. The supernatant was kept; 20 µl of protein A agarose was added to 1 ml of samples and incubated at 4 °C for 1 h.
For protein extraction, the bacterial lysates were incubated overnight with 100 µl of a 50% slurry of glutathione sepharose beads at 4 °C for pre-coupling. The beads were washed 3 times with PBS 1 × and the protein quantity assessed via a Bradford protein assay. HeLa or HEK-293 T cell lysates were then prepared as described above and pre-cleared using 50 µl of glutathione sepharose beads and 25 µg of GST protein for 2 h at 4 °C with end-over-end mixing.
The validation of a predicted interaction with the complement component 1q subcomponent binding protein was done via Western blot as described above , was diluted in a PBS + 0.05% tween-20 solution, and secondary antibody goat anti-mouse IgG coupled to the horseradish peroxidase was diluted in a PBS + 0.05% tween-20 solution) .HeLa or HEK-293 T cells were treated with complete synthetic MTALTND4 or water in DMEM low glucose buffer for 24, 48, and 72 h.
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