Currently I am trying abcam protocol for it.i checked the cell lysis by trypan blue stain which is more than 90 %. but I am facing following issues
1. unable to remove beta actin in mitochondrial fraction.
2. I am able to see mitochondria pellet (cell-1-1.5 X 10^7 cells) but the protein yield is very low 30 ug. and band of interest-SDHA is also very less on western blot compared to whole cell lysate when same amount of total protein (30 ug)
Junhui Sun
Try to pellet down and resuspend into RIPA lysis and extraction buffer (ThermoFisher Sci cat#89900) for Western Blot.
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