Product nameCCCP, Mitochondrial oxidative phosphorylation uncoupler
DescriptionPotent mitochondrial oxidative phosphorylation uncoupler
Biological descriptionPotent mitochondrial oxidative phosphorylation uncoupler. Renders mitochondrial inner membrane permeable to protons. Induces apoptosis in vitro.
Storage instructionsStore at -20°C. Store under desiccating conditions. The product can be stored for up to 12 months.
Solubility overviewSoluble in DMSO to 100 mM and in ethanol to 100 mM
Wherever possible, you should prepare and use solutions on the same day. However, if you need to make up stock solutions in advance, we recommend that you store the solution as aliquots in tightly sealed vials at -20°C. Generally, these will be useable for up to one month. Before use, and prior to opening the vial we recommend that you allow your product to equilibrate to room temperature for at least 1 hour.
Toxic, refer to SDS for further information.
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Our Abpromise guarantee covers the use of ab141229 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Functional Studies||Use at an assay dependent concentration.|
Immunofluorescent analysis of 4 % paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma)(+/- treatment with 10μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP, ab141229) for 24 hours) cells labeling PINK1 with ab216144 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cells treated with 10μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP, ab141229) for 24 hours. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red).
The negative controls are as follows:
-ve control: PBS, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.
All lanes : Anti-PINK1 antibody [EPR20730] (ab216144) at 1/1000 dilution
Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa cells (treated with 10uM carbonyl cyanide 3-chlorophenylhydrazone (CCCP, ab141229) for 24 hours) whole cell lysate
Lysates/proteins at 20 µg per lane.
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Developed using the ECL technique.
Observed band size: 62 kDa why is the actual band size different from the predicted?
Exposure time: 5 seconds
Blocking and dilution buffer: 5% NFDM/TBST
PINK1 can be induced by CCCP treatment (PMID: 24184327).
PINK1 was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) (treated with 10uM carbonyl cyanide 3-chlorophenylhydrazone (CCCP. ab141229) for 24 hours) whole cell lysate with ab216144 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab216144 at 1/500 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366), was used as secondary antibody at 1/1000 dilution.
Lane 1: HeLa (CCCP-treated, ab141229) lysate 10 μg (Input).
Lane 2: ab216144 IP in HeLa (CCCP-treated, ab141229) lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab216144 in HeLa (CCCP-treated, ab141229) whole cell lysate.
Blocking and dilution buffer: 5% NFDM/TBST.
MCF7 cells were incubated at 37°C for 2 hours with vehicle control (0 μM) and different concentrations of CCCP (ab 141229). Increased expression of AKT1 (phospho S473) (ab81283) in MCF7 cells correlates with an increase in CCCP concentration, as described in literature.
Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10 μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab81283 at 2 μg/ml and ab8227 at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 and visualised using ECL development solution.
This product has been referenced in:
- Chaudhari AA et al. Mitochondrial transmembrane potential and reactive oxygen species generation regulate the enhanced effect of CCCP on TRAIL-induced SNU-638 cell apoptosis. J Vet Med Sci 70:537-42 (2008). Read more (PubMed: 18628592) »
- Hirose S et al. Disruption of charge separation followed by that of the proton gradient in the mitochondrial membrane by CCCP. J Biochem 76:213-6 (1974). Read more (PubMed: 4436269) »
- HEYTLER PG & PRICHARD WW A new class of uncoupling agents--carbonyl cyanide phenylhydrazones. Biochem Biophys Res Commun 7:272-5 (1962). Read more (PubMed: 13907155) »