All lanes : Anti-HCE antibody [EPR19384] (ab201046) at 1/2000 dilution

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

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 69 kDa
Observed band size: 69 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-HCE antibody [EPR19384] (ab201046) at 1/2000 dilution

Lane 1 : Ramos (Human Burkitt's lymphoma cell line) whole cell lysate
Lane 2 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 3 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate
Lane 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) 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

Predicted band size: 69 kDa
Observed band size: 69 kDa


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-HCE antibody [EPR19384] (ab201046) at 1/2000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse kidney lysate
Lane 3 : Mouse spleen lysate
Lane 4 : Rat brain lysate
Lane 5 : Rat kidney 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: 69 kDa
Observed band size: 69 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-HCE antibody [EPR19384] (ab201046) at 1/2000 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 1/100000 dilution

Predicted band size: 69 kDa
Observed band size: 69 kDa


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Mouse cerebral cortex tissue labeling HCE with ab201046 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nucleus staining on neurons of mouse cerebral cortex 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 spleen tissue labeling HCE with ab201046 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nucleus staining on lymphocytes of rat spleen 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 cell line from cervix adenocarcinoma) cells labeling HCE with ab201046 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [EPR19384] - 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: ab201046 at 1/500 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 NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling HCE with ab201046 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [EPR19384] - 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: ab201046 at 1/500 dilution followed by ab150120 at 1/1000 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

HCE was immunoprecipitated from 1mg of SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate with ab201046 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab201046 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: SH-SY5Y whole cell lysate 10µg (Input).
Lane 2: ab201046 IP in SH-SY5Y whole cell lysate.
Lane 3: Rabbit IgG,monoclonal [EPR19384] -Isotype Control (ab172730) instead of ab201046 in SH-SY5Y whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.