Anti-p73 antibody [EPR18409(T)(MIX)] (ab189896) at 1/5000 dilution + Recombinant fragment of human p73 protein at 0.005 µg

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

Predicted band size: 70 kDa
Observed band size: 28 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Recombinant fragment of human p73 protein contains aa380-636 with His-Tag®.

All lanes : Anti-p73 antibody [EPR18409(T)(MIX)] (ab189896) at 1/5000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 3 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate
Lane 4 : WEHI-3 (Mouse leukemia cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 70 kDa
Observed band size: 70 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-p73 antibody [EPR18409(T)(MIX)] (ab189896) at 1/5000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/50000 dilution

Predicted band size: 70 kDa
Observed band size: 70 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-p73 antibody [EPR18409(T)(MIX)] (ab189896) at 1/5000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse heart lysate
Lane 3 : Mouse kidney lysate
Lane 4 : Mouse spleen lysate
Lane 5 : Rat brain lysate
Lane 6 : Rat spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 70 kDa
Observed band size: 70 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-p73 antibody [EPR18409(T)(MIX)] (ab189896) at 1/5000 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
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 4 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 70 kDa
Observed band size: 70 kDa


Exposure time: 5 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human cerebral cortex tissue labeling p73 alpha+beta with ab189896 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Negative staining on Human cerebral cortex.

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 testis tissue labeling p73 alpha+beta with ab189896 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasmic and weak nucleus staining on Human spermatogonial cells 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 glioma tissue labeling p73 alpha+beta with ab189896 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus and cytoplasmic staining on Human glioma 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 gastric carcinoma tissue labeling p73 alpha+beta with ab189896 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus and cytoplasmic staining on Human gastric carcinoma 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 MCF7 (Human breast adenocarcinoma cell line) cells labeling p73 alpha+beta with ab189896 at 1/1000 dilution, followed by Goat anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear and cytoplasm staining on MCF7 cell line. 

The nuclear counterstain is DAPI (blue).

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

The negative controls are as follows:-

-ve control 1: ab189896 at 1/1000 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2: ab7291at 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 p73 alpha+beta with ab189896 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

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

The nuclear counterstain is DAPI (blue).

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

The negative controls are as follows:-

-ve control 1: ab189896 at 1/1000 dilution followed by ab150120 at 1/1000 dilution.

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

Flow cytometric analysis of 4% paraformaldehyde-fixed Jurkat (Human T cell leukemia cell line from peripheral blood) cells labeling p73 alpha+beta with ab189896 at 1/300 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 1/500 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde-fixed K562 (Human chronic myelogenous leukemia cell line from bone marrow) cells labeling p73 alpha+beta with ab189896 at 1/300 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 1/500 dilution was used as the secondary antibody.

p73 alpha+beta was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab189896 at 1/80 dilution.

Western blot was performed from the immunoprecipitate using ab189896 at 1/1000 dilution.

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

Lane 1: HeLa whole cell lysate 10ug (Input).

Lane 2: ab189896 IP in HeLa whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab189896 in HeLa whole cell lysate.

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

Exposure time: 10 seconds.

p73 alpha+beta was immunoprecipitated from 1mg of MCF7 (Human breast adenocarcinoma cell line) whole cell lysate with ab189896 at 1/80 dilution.

Western blot was performed from the immunoprecipitate using ab189896 at 1/1000 dilution.

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

Lane 1: MCF7 whole cell lysate 10ug (Input).

Lane 2: ab189896 IP in MCF7 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab189896 in MCF7 whole cell lysate.

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

Exposure time: 10 seconds.