All lanes : Anti-MTCO1 antibody [EPR19628] (ab203912) at 1/2000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) mitochondria lysate
Lane 3 : Neuro-2a (Mouse neuroblastoma cell line) whole cell lysate
Lane 4 : Neuro-2a (Mouse neuroblastoma cell line) mitochondria lysate
Lane 5 : Mouse liver whole cell lysate
Lane 6 : Mouse liver mitochondria lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 57 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane1: 3 minutes; Lane 2: 30seconds; Lane 3 and 4: 10seconds; Lane 5: 3 minutes; Lane 6: 1 minute.

The molecular weight  observed is consistent with what has been described in the literature (PMID: 23125210 & 22426402).

All lanes : Anti-MTCO1 antibody [EPR19628] (ab203912) at 1/1000 dilution

Lane 1 : C6 (Rat glial tumor cell line) mitochondria lysate
Lane 2 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) mitochondria lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 57 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-MTCO1 antibody [EPR19628] (ab203912) at 1/1000 dilution + Human fetal liver whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 57 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-MTCO1 antibody [EPR19628] (ab203912) at 1/1000 dilution

Lane 1 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 57 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human cardiac muscle tissue labeling MTCO1 with ab203912 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on Human cardiac muscle is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human thyroid cancer tissue labeling MTCO1 with ab203912 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on Human thyroid cancer is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse cardiac muscle tissue labeling MTCO1 with ab203912 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on mouse cardiac muscle is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat cardiac muscle tissue labeling MTCO1 with ab203912 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on rat cardiac muscle is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling MTCO1 with ab203912 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on HeLa cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab203912 at 1/250 dilution followed by ab150120  at 1/1000 dilution.

-ve control 2: ab7291 at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling MTCO1 with ab203912 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on HeLa cell line.

The nuclear counterstain is DAPI (blue).

COX IV is detected with ab33985 (anti-COX IV mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor^®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab203912 at 1/250 dilution followed by ab150120  at 1/1000 dilution.

-ve control 2: ab33985  at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Neuro-2a (Mouse neuroblastoma cell line) cells labeling MTCO1 with ab203912 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on Neuro-2a cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab203912 at 1/250 dilution followed by ab150120  at 1/1000 dilution.

-ve control 2: ab7291  at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Neuro-2a (Mouse neuroblastoma cell line) cells labeling MTCO1 with ab203912 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on Neuro-2a cell line.

The nuclear counterstain is DAPI (blue).

COX IV is detected with ab33985 (anti-COX IV mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor^®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab203912 at 1/250 dilution followed by ab150120  at 1/1000 dilution.

-ve control 2: ab33985 at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling MTCO1 with ab203912 at 1/70 dilution (red) compared with a Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor® 488) at 1/500 dilution was used as the secondary antibody.

Cells were permeabilised with 90% methanol (diluted with 1X PBS).