Following siRNA mediated knockdown, U2OS cells were collected and lysed for 10 min on ice in lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% TX-100 and 2.5 mM PMSF) followed by a centrifugation step of 14 000 × g for 5 min at 4°C. 60 μg of cellular lysates were separated by SDS-PAGE followed by western blotting onto a supported nitrocellulose membranes. Membranes were probed with antibodies against proteins of interest and HRP-conjugated secondary antibodies followed by ECL detection. ECL reactions were visualized with a ChemiDoc instrument (Bio Rad). Antibodies used for Western blot detection were mtSSB (Sigma, HPA002866); TFAM (kind gift of Dr R. Wiesner); Twinkle (mouse-monoclonal, kind gift from Prof. Anu Suomalainen-Wartiovaara; see also (1 (link))); CoxII (Abcam, ab110258); PHB1 (Abcam, ab28172); MRPL3 (Abcam, ab39268); MRPS22 (ProteinTech, 10984-AP); MRPL49 (ProteinTech, 15542-AP), GRSF1 (Sigma, HPA036985), Suv3 (kind gift of Dr Roman Szczesny), Actin (Novusbio, NB600-532H).
Protocol detail
Characterization of Mitochondrial Protein Levels
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Actin
Antibodies
Buffer
Cellular
Centrifugation
Coxii
Edta
Membranes
Mouse
Nacl
Nitrocellulose
Proteins
Sds page
Sirna
Tfam
Tris
Tx 100
Western blot
Corresponding Organization : Institut de Biologia Molecular de Barcelona
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Variable analysis
independent variables
- SiRNA mediated knockdown
- Protein expression levels of mtSSB, TFAM, Twinkle, CoxII, PHB1, MRPL3, MRPS22, MRPL49, GRSF1, Suv3, Actin
- U2OS cells
- Lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% TX-100 and 2.5 mM PMSF)
- Centrifugation step of 14 000 × g for 5 min at 4°C
- 60 μg of cellular lysates
- SDS-PAGE
- Western blotting onto supported nitrocellulose membranes
- Antibodies used for Western blot detection: mtSSB, TFAM, Twinkle, CoxII, PHB1, MRPL3, MRPS22, MRPL49, GRSF1, Suv3, Actin
- None specified
- None specified
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