All lanes : Anti-BTF3 antibody [EPR16495] (ab203517) at 1/2000 dilution

Lane 1 : MCF7 (Human breast adenocarcinoma cell line) cell lysate
Lane 2 : SK-BR-3 (Human mammary gland adenocarcinoma cell line) cell lysate
Lane 3 : HeLa (Human epithelial cells from cervix adenocarcinoma) cell lysate
Lane 4 : Jurkat (Human T cell leukemia cells from peripheral blood) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

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


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-BTF3 antibody [EPR16495] (ab203517) at 1/1000 dilution + Human fetal kidney lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

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


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-BTF3 antibody [EPR16495] (ab203517) at 1/1000 dilution + C6 (Rat glial tumor cells) cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

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


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-BTF3 antibody [EPR16495] (ab203517) at 1/1000 dilution + Human lymph node lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 22 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-BTF3 antibody [EPR16495] (ab203517) at 1/1000 dilution

Lane 1 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) cell lysate
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma) cell lysate
Lane 3 : NIH/3T3 (Mouse embyro fibroblast cells) cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 22 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

All lanes : Anti-BTF3 antibody [EPR16495] (ab203517) at 1/1000 dilution

Lane 1 : Mouse heart lysate
Lane 2 : Mouse spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 22 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma labeling BTF3 with ab203517 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasmic staining on Human cervix carcinoma tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (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 spleen labeling BTF3 with ab203517 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasmic staining on Human spleen tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (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 liver tissue labeling BTF3 with ab203517 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasmic staining on mouse liver tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (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 testis tissue labeling BTF3 with ab203517 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasmic staining on rat testis tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (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 cells from cervix adenocarcinoma) cells labeling BTF3 with ab203517 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal imaging showing cytoplasmic and weakly nuclear staining on HeLa cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin 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: ab203517 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SK-BR-3 (Human mammary gland adenocarcinoma cell line) cells labeling BTF3 with ab203517 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal imaging showing cytoplasmic and weakly nuclear staining on SK-BR-3 cell line. 

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin 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: ab203517 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling BTF3 with ab203517 at 1/250 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

BTF3 was immunoprecipitated from 1mg of NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate with ab203517 at 1/70 dilution.

Western blot was performed from the immunoprecipitate using ab203517 at 1/1000 dilution.

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: NIH/3T3 whole cell lysate 10ug (Input).

Lane 2: ab203517 IP in NIH/3T3 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab203517 in NIH/3T3 whole cell lysate.

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

Exposure time: 10 seconds.