重组Anti-Heme Oxygenase 1抗体[EPR18161-128] (ab189491)

False colour image of Western blot: Anti-Heme Oxygenase 1 antibody [EPR18161-128] staining at 1/2000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab189491 was shown to bind specifically to Heme Oxygenase 1. A band was observed at 32 kDa in wild-type A549 cell lysates with no signal observed at this size in HMOX1 knockout cell line ab269503 (HMOX knockout A549 lysate ab259782).

To generate this image, wild-type and HMOX1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween®20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

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).

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).

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).

Intracellular flow cytometric analysis of 90% methanol/ 4% paraformaldehyde fixed NIH/3T3 (mouse embryonic fibroblast) cell line labeling Heme Oxygenase1 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 Fluorr^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody. 

Intracellular flow cytometric analysis of 90% methanol/4% paraformaldehyde fixed HeLa (human cervix adenocarcinoma epithelial cell) cell line labeling Heme Oxygenase1 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 Fluorr^® 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).