Cell lysates were prepared using RIPA buffer (see lysis and click chemistry methods below) and run on SDS Tris-Glycine gels (Precast 4 – 20% Bio-Rad, or hand-casted 20%). Gels were transferred to low-background PVDF membranes using the BioRad TransBlot Turbo system and blocked with 5% milk powder in TBS + 0.1% Tween 20 (TBST) for 1 hr at room temperature. Primary antibodies were diluted at 1:1000 unless otherwise specified in 5% BSA in TBST + 0.1% sodium azide and incubated overnight at 4 °C. Membranes were washed for 5 min x 5 with TBST, and appropriate Starbright Blue 700 secondary antibody (Bio-Rad, Hercules, CA) for 1 hr at room temperature, followed by 5 × 5 min washes with TBST. Membranes were imaged using the ChemiDoc MP imaging system. Antibodies used for immunoblotting were: ATP5B (Proteintech, 17247–1AP), VCAC1 (CST #4661), TIMM17B (Proteintech 11062–1-AP), FLAG® (Sigma Aldrich, M2), ATP5G1 (Abcam, EPR13908), Actin (BioRad, 12004163).
Timm17b
Manufactured by Proteintech
TIMM17B is a mitochondrial protein that plays a role in the import of proteins into the mitochondrial inner membrane. It is part of the TIM23 complex, which is responsible for the translocation of proteins across the mitochondrial inner membrane.
Automatically generated - may contain errors
2 protocols using timm17b
1
Western Blot Analysis of Mitochondrial Proteins
2
Western Blot Analysis of Mitochondrial Proteins
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Cell lysates were prepared using RIPA buffer (see lysis and click chemistry methods below) and run on SDS Tris-Glycine gels (Precast 4 – 20% Bio-Rad, or hand-casted 20%). Gels were transferred to low-background PVDF membranes using the BioRad TransBlot Turbo system and blocked with 5% milk powder in TBS + 0.1% Tween 20 (TBST) for 1 hr at room temperature. Primary antibodies were diluted at 1:1000 unless otherwise specified in 5% BSA in TBST + 0.1% sodium azide and incubated overnight at 4 °C. Membranes were washed for 5 min x 5 with TBST, and appropriate Starbright Blue 700 secondary antibody (Bio-Rad, Hercules, CA) for 1 hr at room temperature, followed by 5 × 5 min washes with TBST. Membranes were imaged using the ChemiDoc MP imaging system. Antibodies used for immunoblotting were: ATP5B (Proteintech, 17247–1AP), VCAC1 (CST #4661), TIMM17B (Proteintech 11062–1-AP), FLAG® (Sigma Aldrich, M2), ATP5G1 (Abcam, EPR13908), Actin (BioRad, 12004163).
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