Immunohistochemical analysis of paraffin embedded mouse liver tissue labeling Heme Oxygenase 1 with ab189491 at 1/20000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining on Kupffer cells of mouse liver (PMID: 9449694) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Anti-Heme Oxygenase 1 antibody [EPR18161-128] (ab189491) at 1/2000 dilution + Human spleen lysate at 10 µg

Secondary
VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution

Predicted band size: 33 kDa
Observed band size: 28, 32 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID: 18400743).

The lower band observed is a truncated form of Heme Oxygenase 1 (PMID: 17430897).

Immunofluorescent analysis of 4% paraformaldehyde fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Heme Oxygenase 1 with ab189491 at 1/250 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cells. Details of counterstains: ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution; DAPI for nuclei.
The negative controls are as follows: Secondary antibody only for control.

Immunofluorescent analysis of 4% paraformaldehyde fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeling Heme Oxygenase 1 with ab189491 at 1/250 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on NIH/3T3 cells. Details of counterstains: ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution; DAPI for nuclei.
The negative controls are as follows: Secondary antibody only for control.

Immunohistochemical analysis of paraffin embedded human liver tissue labeling Heme Oxygenase 1 with ab189491 at 1/20,000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining on Kupffer cells of human liver (PMID: 9449694) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin embedded rat liver tissue labeling Heme Oxygenase 1 with ab189491 at 1/20,000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining on Kupffer cells of rat liver (PMID: 9449694) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

All lanes : Anti-Heme Oxygenase 1 antibody [EPR18161-128] (ab189491) at 1/5000 dilution

Lane 1 : NIH/3T3 (mouse embryonic fibroblast) lysate
Lane 2 : Rat spleen lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell) lysate
Lane 4 : Mouse spleen 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: 33 kDa
Observed band size: 28, 32 kDa why is the actual band size different from the predicted?

Blocking: 5% NFDM/TBST.

Exposure time:

Lanes 1,2 and 3: 2 seconds;

Lane 4: 1 second

The molecular weight observed is consistent with what has been described in the literature (PMID: 18400743).

The lower band observed is a truncated form of Heme Oxygenase 1 (PMID: 17430897).

Flow cytometric analysis of 90% methanol/ 4% paraformaldehyde fixed NIH/3T3 (mouse embryonic fibroblast) cell line labeling Heme Oxygenase 1  with ab189491 at 1/50 (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 90% methanol/4% paraformaldehyde fixed HeLa (human cervix adenocarcinoma epithelial cell) cell line labeling Heme Oxygenase 1  with ab189491 at 1/50 (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

Heme Oxygenase 1 was immunoprecipitated from 0.35 mg of NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab189491 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab189491 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.
Lane 1: NIH/3T3 (mouse embryonic fibroblast) whole cell lysate  10 μg (Input).
Lane 2: NIH/3T3 whole cell lysate (+).
Lane 3: Rabbit monoclonal IgG (ab172730) instead of  ab189491 in NIH/3T3 whole cell lysate (-).
Blocking and dilution buffer and concentration: 5% NFDM/TBST.

The truncated form of Heme Oxygenase 1 is described in the literature (PMID: 17430897).