Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HT-29 (Human colorectal adenocarcinoma cells) cells labeling CYP2D6 with ab185625 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Cytoplasmic staining on HT-29 cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab185625 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

 

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling CYP2D6 with ab185625 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Cytoplasmic staining on Human liver is observed. Counter stained with Hematoxylin.

Negative 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 kidney tissue labeling CYP2D6 with ab185625 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Cytoplasmic staining on Human kidney is observed. Counter stained with Hematoxylin.

Negative 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 hepatocellular carcinoma tissue labeling CYP2D6 with ab185625 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Cytoplasmic staining on Human hepatocellular carcinoma is observed. Counter stained with Hematoxylin.

Negative 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.

Flow cytometric analysis of 2% paraformaldehyde-fixed HT-29 (Human colorectal adenocarcinoma cells) cells labeling CYP2D6 with ab185625 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 1/150 dilution was used as the secondary antibody.

All lanes : Anti-CYP2D6 antibody [EPR17868] (ab185625) at 1/10000 dilution

Lane 1 : Full-length CYP2D6 transfected 293T lysate
Lane 2 : Non-transfected 293T lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 55, 50 kDa
Observed band size: 81 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

 

Full-length CYP2D6 293T overexpressed lysates contain GFP tagged-aa1-497.

Anti-CYP2D6 antibody [EPR17868] (ab185625) at 1/10000 dilution + Human fetal liver lysate at 20 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 55, 50 kDa
Observed band size: 50,55 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Based on the sequence analysis, ab185625 recognizes two isoforms with the predicted MWs of 55kDa and 50kDa respectively.

All lanes : Anti-CYP2D6 antibody [EPR17868] (ab185625) at 1/10000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate
Lane 3 : HepG2 (Human liver hepatocellular carcinoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 55, 50 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-CYP2D6 antibody [EPR17868] (ab185625) at 1/1000 dilution + HT-29 (Human colorectal adenocarcinoma cells) whole cell lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 55, 50 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.