Product nameHeLa Mitochondrial DNA Depletion Cell Lysate Set: Ethidium Bromide-Treated and Vehicle-Treated
HeLa cells were cultured in the presence or absence of 50 ng/mL Ethidium Bromide for 14 days. Whole cell lysates were prepared in an SDS lysis buffer containing protease and phosphatase inhibitors. Ethidium bromide cells are judged to be depleted of mitochondrial DNA (termed Rho0) on the basis of western blotting.
Concentration: HeLa Rho0 lysate, 200 µg at 1.0 mg/mL
HeLa control lysate, 200 µg at 1.0 mg/mL
Human mitochondrial DNA (mtDNA) encodes 13 subunits of the oxidative phosphorylation (OXPHOS) enzyme complexes I, III, IV and V. Low concentrations of ethidium bromide inhibit mtDNA synthesis without having overtly effecting cell viability. Loss of mtDNA not only depletes mtDNA encoded proteins but also can lead to decreased levels of nuclear DNA encoded mitochondrial proteins due to protein complex instabilities
Tested applicationsSuitable for: WB, SDS-PAGEmore details
Storage instructionsStore at -80°C. Please refer to protocols.
Storage bufferConstituents: 0.03% Sodium fluoride, 0.01% Bromophenol blue, 1.8% SDS, 0.77% DTT, 1.1% Tris, 10% Glycerol, 0.7% Sodium chloride
Components 2 units HeLa control lysate 1 x 200µg HeLa RhoO lysate 1 x 200µg
Our Abpromise guarantee covers the use of ab154479 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use at an assay dependent concentration.|
|SDS-PAGE||Use at an assay dependent concentration. Customer may choose to boil the sample before loading, at their discretion.|
Western blot of HeLa Whole Cell Lysate: Rho treated and control (ab154479) using MitoProfile® Total OXPHOS Human WB Antibody Cocktail (ab110411). The results show a partial or complete reduction of subunits across the five OXPHOS complexes 1-V.
Lane 1 HeLa control lysate, 15µg
Lane 2 HeLa Rho0 lysate, 15µg
Block with 4% milk/PBS
Primary antibody: MitoProfile® Total OXPHOS Human WB Antibody Cocktail (ab110411), 1/208.
Secondary antibody: anti-mouse-HRP, 1/5000
Coomassie blue stained SDS-PAGE gel of HeLa Control and Rho0 lysates (ab154479) to demonstrate equivalent protein normalization.
Lane1. MW Markers
Lane2. HeLa control lysate (ab154479); 20µg load
Lane3. HeLa Rho0 lysate (ab154479); 20µg load
ab154479 has not yet been referenced specifically in any publications.