Western Blotting Analysis of Mitochondrial Proteins

Western blotting analysis was carried out as detailed previously (23 (link), 42 (link)). 20 micrograms of total proteins obtained from lysed mitochondria were denatured and loaded on sodium dodecyl sulfate (SDS) polyacrylamide gels. The gels were electroblotted onto a polyvinylidene difluoride (PVDF) membrane for hybridization. The antibodies used for this investigation were from Abcam [ND1 (ab74257), ND3 (ab170681), ND6 (ab81212), TOM20 (ab56783), Total OXPHOS Human WB Antibody Cocktail (ab110411) and P62 (ab56416) ], Proteintech Group [CYTB (55090-1-AP), CO2 (55070-1-AP), ATP8 (26723-1-AP), CLPP (15698-A-AP), LONP1 (15440-1-AP), OPA1 (27733-1-AP), FIS1 (10956-1-AP), MFN1 (13798-1-AP), MFF (17090-1-AP), MFN2 (12186-1-AP) and DRP1 (12957-1-AP)], ABclonal Technology [β-Actin (AC026), ND4 (A9941), ND5 (A17972), CO1 (A17889), CO3 (A17891), LC3 (A19665), Parkin (A0968) and Cyt C (A4912)], Cell Signaling [Autophagy Antibody Sampler Kit (4445) and Apoptosis Antibody Sampler Kit (9915)] and Abcepta [PINK1 (AW5456)]. Peroxidase AffiniPure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as secondary antibodies and protein signals were detected using the ECL system (CW-BIO). Quantification of density in each band was performed as detailed elsewhere (42 (link)).

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Chen X., Meng F., Chen C., Li S., Chou Z., Xu B., Mo J.Q., Guo Y, & Guan M.X. (2024). Deafness-associated tRNAPhe mutation impaired mitochondrial and cellular integrity. The Journal of Biological Chemistry, 300(5), 107235.

Publication 2024

Total OXPHOS Human WB Antibody Cocktail β-Actin Peroxidase AffiniPure goat anti-mouse IgG goat anti-rabbit IgG ECL system

Corresponding Organization : Second Affiliated Hospital of Zhejiang University

Other organizations : Lanzhou University, First Hospital of Lanzhou University, Gansu Provincial Hospital, Lanzhou University Second Hospital, Rady Children's Hospital-San Diego

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Variable analysis

independent variables

Primary antibodies used for Western blotting analysis

dependent variables

Protein expression levels of mitochondrial proteins (ND1, ND3, ND6, CYTB, CO2, ATP8, CO1, CO3, CLPP, LONP1, OPA1, FIS1, MFN1, MFN2, DRP1)
Protein expression levels of autophagy and apoptosis-related proteins (P62, LC3, Parkin, Cyt C)

control variables

Total protein amount (20 micrograms) loaded on SDS-polyacrylamide gels
Lysed mitochondrial samples used as the source of proteins

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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