CCCP, Mitochondrial oxidative phosphorylation uncoupler

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - CCCP, Mitochondrial oxidative phosphorylation uncoupler (AB141229)

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.

• FuncS

Unknown

Functional Studies - CCCP, Mitochondrial oxidative phosphorylation uncoupler (AB141229)

MCF7 cells were incubated at 37°C for 2 hours with vehicle control (0 μM) and different concentrations of CCCP (ab141229). 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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - CCCP, Mitochondrial oxidative phosphorylation uncoupler (AB141229)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PC-3 (human prostate adenocarcinoma epithelial cell) cells labelling Ubiquitin (phospho S65) with ab320096 at 1/50 (18.68 ug/ml) dilution, followed by ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing increased staining in PC-3 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195884 Anti-Tubulin rat monoclonal antibody - Microtubule Marker (Alexa Fluor® 647) was used to counterstain tubulin at 1/100 5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) at 1/1000 2ug/ml dilution.

• WB

Supplier Data

Western blot - CCCP, Mitochondrial oxidative phosphorylation uncoupler (AB141229)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 22915595 and PMID : 30250224).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-PGAM5 antibody [RM1254] (<a href='/en-us/products/primary-antibodies/pgam5-antibody-rm1254-ab322207'>ab322207</a>) at 1/1000 dilution

Lane 1:

Untreated PANC-1(human pancreatic epithelioid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

PANC-1 treated with 100 uM CCCP (ab141229) for 2 hours, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 32 kDa,36 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - CCCP, Mitochondrial oxidative phosphorylation uncoupler (AB141229)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Ubiquitin antibody [EPR8830] (ab134953) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) (<a href='/en-us/products/primary-antibodies/ubiquitin-phospho-s65-antibody-30h3-30k1-rat-igg2a-chimeric-ab320096'>ab320096</a>) at 1/1000 dilution

Lane 1:

Untreated PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

PC-3 treated with 30 μM CCCP (ab141229) for 2 hours, whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Lane 4:

PC-3 treated with 30 μM CCCP (ab141229) for 2 hours, whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 8-200 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - CCCP, Mitochondrial oxidative phosphorylation uncoupler (AB141229)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Ubiquitin antibody [EPR8830] (ab134953) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) (<a href='/en-us/products/primary-antibodies/ubiquitin-phospho-s65-antibody-30h3-30k1-rat-igg2a-chimeric-ab320096'>ab320096</a>) at 1/1000 dilution

Lane 1:

Untreated Wild-type 293T (human embryonic kidney epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

293T treated with 20 μM CCCP (ab141229) for 8 hours, whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated PINK1 knockout HEK-293T whole cell lysate (untreated membrane) at 20 µg

Lane 4:

PINK1 knockout HEK-293T treated with 20 μM CCCP (ab141229) for 8 hours, whole cell lysate (untreated membrane) at 20 µg

Lane 5:

Untreated Wild-type 293T (human embryonic kidney epithelial cell) whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Lane 6:

293T treated with 20 μM CCCP (ab141229) for 8 hours, whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Lane 7:

Untreated PINK1 knockout HEK-293T whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Lane 8:

PINK1 knockout HEK-293T treated with 20 μM CCCP (ab141229) for 8 hours, whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 8-200 kDa,36 kDa

false

Exposure time: 92s

• IP

Supplier Data

Immunoprecipitation - CCCP, Mitochondrial oxidative phosphorylation uncoupler (AB141229)

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 Detection Reagent (HRP) (ab131366), was used for detection 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.

true

Exposure time: 5s

• Chemical Structure

Lab

Chemical Structure - CCCP, Mitochondrial oxidative phosphorylation uncoupler (AB141229)

2D chemical structure image of ab141229, CCCP, Mitochondrial oxidative phosphorylation uncoupler