Protein Fractionation and Immunodetection Protocol
Protein fractionation, transfer and immuno-detection were performed as described (Dalla Rosa et al., 2014 (link)), with some modifications. Muscle and liver samples were prepared as previously described (Cooper et al., 2003 (link)). Cells were lysed on ice in phosphate-buffered saline (PBS), 0.1% n-dodecyl β-d-maltoside (DDM), 1% SDS, 1 × protease inhibitor cocktail (Roche), 50 U Benzonase® and phosphatase inhibitor complexes (Cell signalling), or in RIPA buffer containing 1× protease inhibitor cocktail (Roche). Protein concentration was measured by Lowry assay (DC™ Reagent, Bio-Rad) or BCA assay, and 10 μg of lysate analysed per lane. Primary antibodies were: mouse anti-GAPDH (1:20 000, Abcam), mouse anti-NDUFB8 (1:1000, Abcam), mouse anti-COX II (1:2000, Abcam), mouse anti-VDAC1 (Porin) (1:10000, Merck), rabbit anti-ATAD3 (1:60 000, gift from John Walker), rabbit anti-SREBF2 (1:1000, Abcam), rabbit anti-CES-1 (1:2000, Proteintech).
Desai R., Frazier A.E., Durigon R., Patel H., Jones A.W., Dalla Rosa I., Lake N.J., Compton A.G., Mountford H.S., Tucker E.J., Mitchell A.L., Jackson D., Sesay A., Di Re M., van den Heuvel L.P., Burke D., Francis D., Lunke S., McGillivray G., Mandelstam S., Mochel F., Keren B., Jardel C., Turner A.M., Ian Andrews P., Smeitink J., Spelbrink J.N., Heales S.J., Kohda M., Ohtake A., Murayama K., Okazaki Y., Lombès A., Holt I.J., Thorburn D.R, & Spinazzola A. (2017). ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism. Brain, 140(6), 1595-1610.
Antibodies AssayBenzonase BufferCells Cox ii Fractionation GapdhLiver Mouse Muscle Phosphatase PhosphatePorin Protease inhibitor Protein Rabbit RipaRosa Saline Vdac1 Walker
Corresponding Organization :
Other organizations :
Royal Children's Hospital, Murdoch Children's Research Institute, University College London, The Royal Free Hospital, The Francis Crick Institute, MRC Mitochondrial Biology Unit, Radboud University Nijmegen, Radboud University Medical Center, Great Ormond Street Hospital, Victorian Clinical Genetics Services, University of Melbourne, Florey Institute of Neuroscience and Mental Health, Pitié-Salpêtrière Hospital, Sorbonne Université, Assistance Publique – Hôpitaux de Paris, Inserm, Centre National de la Recherche Scientifique, International Council on Mining and Metals, Institut Cochin, Université Paris Cité, UNSW Sydney, Sydney Children's Hospital, National Hospital for Neurology and Neurosurgery, Saitama Medical University, Chiba Hospital, Ikerbasque, Biogipuzkoa Health Research Institute
Protein concentration measurement by Lowry assay (DC™ Reagent, Bio-Rad) or BCA assay
10 μg of lysate analyzed per lane
positive controls
None specified
negative controls
None specified
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