重组Anti-Total OXPHOS抗体Cocktail抗体[RM1134] - BSA and Azide free (ab317271)

All lanes : Anti-Total OXPHOS Antibody Cocktail antibody [RM1134] (ab317270) at 1/1000 dilution

Lane 1 : HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate
Lane 3 : HEK-293 (human embryonic kidney epithelial cell) whole cell lysate
Lane 4 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 5 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 18,22,29,48,54 kDa why is the actual band size different from the predicted?

This data was developed using ab317270, the same antibody clone in a different buffer formulation.

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

Exposure time: Lanes 1-3: 15 seconds; Lanes 4-5: 37 seconds

All lanes : Anti-Total OXPHOS Antibody Cocktail antibody [RM1134] (ab317270) at 1/1000 dilution

Lane 1 : Human cerebellum tissue lysate
Lane 2 : Human kidney tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 18,22,29,48,54 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

This data was developed using ab317270, the same antibody clone in a different buffer formulation.

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

All lanes : Anti-Total OXPHOS Antibody Cocktail antibody [RM1134] (ab317270) at 1/1000 dilution

Lane 1 : MCF7 non-mitochondrial fraction
Lane 2 : MCF7 mitochondria fraction

Lysates/proteins at 20 µg per lane.

Secondary
Lane 1 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
Lane 2 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 18,22,29,48,54 kDa why is the actual band size different from the predicted?


Exposure time: 8 seconds

This data was developed using ab317270, the same antibody clone in a different buffer formulation.

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-COX IV antibody [EPR9442 (ABC)] - Mitochondrial Loading Control (ab202554) staining at 17kDa dilution.

This data was developed using ab317270, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Total OXPHOS Antibody Cocktail with ab317270 at 1/2000 (0.246 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human liver. The section was incubated with ab317270 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

This data was developed using ab317270, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling Total OXPHOS Antibody Cocktail with ab317270 at 1/2000 (0.246 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human breast carcinoma. The section was incubated with ab317270 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

This data was developed using ab317270, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling Total OXPHOS Antibody Cocktail with ab317270 at 1/500 (0.984 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/mL dilution (Green).

Confocal image showing mitochondrial staining in HepG2 cell line (shown in green). The counter stain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

anti-COX IV mouse monoclonal antibody - Mitochondrial Marker was used to counterstain tubulin at 1/1000 1ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

This data was developed using ab317270, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling Total OXPHOS Antibody Cocktail with ab317270 at 1/500 dilution (0.1ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.