Anti-Heme Oxygenase 1 抗体 [23/Heme Oxygenase 1] (ab302671)

All lanes : Anti-Heme Oxygenase 1 antibody [23/Heme Oxygenase 1] (ab302671) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : A549 (human lung carcinoma epithelial cell), whole cell lysate
Lane 3 : Untreated THP-1 (human monocytic leukemia monocyte), whole cell lysate
Lane 4 : THP-1 treated with 80nm TPA (ab120297) for 24hours, then 1ug/ml LPS for 30 minutes, whole cell lysate
Lane 5 : HT-1080 (human fibrosarcoma epithelial cell), whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

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

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 8247003 ).

The identity of the band(s) between 75kDa~100kDa are unknown.

Lanes 3/4/5 in blot was developed using a high sensitivity ECL substrate.

Exposure time: Lanes 1-5: 3 minutes. 

Immunohistochemical analysis of paraffin-embedded human liver muscle tissue labeling Heme Oxygenase 1 with ab302671 at 1/2000 dilution (0.501 μg/ml), followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on Kupffer cells of human liver was observed. The section was incubated with AB302671 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. 

Secondary antibody only control: Primary diluent was used instead of primary antibody, followed by ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). 

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used. 

Immunohistochemical analysis of paraffin-embedded human lung tissue labeling Heme Oxygenase 1 with ab302671 at 1/2000 dilution (0.501 μg/ml), followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on macrophages of human lung was observed. The section was incubated with AB302671 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. 

Secondary antibody only control: Primary diluent was used instead of primary antibody, followed by ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). 

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used. 

Heme Oxygenase 1 was immunoprecipitated from 0.35 mg A549 (human lung carcinoma epithelial cell) whole cell lysate with ab302671 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab302671 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: A549 (human lung carcinoma epithelial cell) whole cell lysate 10 μg (Inset)

Lane 2: ab302671 IP in A549 whole cell lysate

Lane 3: Mouse IgG1, kappa monoclonal isotype control (ab18443) instead of ab302671 in A549 whole cell lysate

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

Exposure time: 75 seconds.

Observed M_{W (kDa): 32.}