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).
Starbright blue 700 secondary antibody
Manufactured by Bio-Rad
The Starbright Blue 700 secondary antibody is a fluorescently labeled antibody designed for use in immunoassays and imaging applications. It is capable of binding to and detecting primary antibodies from various species.
Automatically generated - may contain errors
2 protocols using starbright blue 700 secondary antibody
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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