Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling Angiotensinogen with ab276132 at 1/500 (0.854 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on rat kidney (PMID: 28716988). The section was incubated with ab276132 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 100% Methanol permeabilized HepG2 cells labelling Angiotensinogen with ab276132 at 1/100 (4.27 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000  dilution (Green). Confocal image showing cytoplasmic staining in HepG2 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

All lanes : Anti-Angiotensinogen antibody [EPR24118-2] (ab276132) at 1/1000 dilution

Lane 1 : HepG2 (human hepatocellar carcinoma epithelial cell), whole cell lysate
Lane 2 : Huh7 (human hepatocellar carcinoma epithelial cell), whole cell lysate
Lane 3 : Human lung tissue lysate
Lane 4 : Human liver tissue lysate
Lane 5 : Human kidney tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 53 kDa
Observed band size: 48-70 kDa why is the actual band size different from the predicted?

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

Angiotensinogen undergoes glycosylation. The molecular weight observed is consistent with what has been described in literature(PMID: 9694881).

Lanes 1 & 2 of the blot were developed using a higher sensitivity ECL substrate.

Exposure times: Lane 1, 2: 59 seconds; Lane 3: 3.25 seconds;Lane 4, 5: 15 seconds

 

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling Angiotensinogen with ab276132 at 1/500 dilution (0.1ug)(Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Angiotensinogen was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate with ab276132 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab276132 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate 10 ug

Lane 2: ab276132 IP in HepG2 whole cell lysate

Lane 3:  Rabbit monoclonal IgG (ab172730) instead of ab276132 in HepG2 whole cell lysate

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

Exposure time: 23 seconds.

 

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling Angiotensinogen with ab276132 at 1/500 (0.854 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human placenta (PMID: 21215447). The section was incubated with ab276132 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

All lanes : Anti-Angiotensinogen antibody [EPR24118-2] (ab276132) at 1/1000 dilution

Lane 1 : Rat lung tissue lysate
Lane 2 : Rat liver tissue lysate
Lane 3 : Rat kidney tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 53 kDa
Observed band size: 48-70 kDa why is the actual band size different from the predicted?

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

Angiotensinogen undergoes glycosylation. The molecular weight observed is consistent with what have been described in literature(PMID: 9694881).

This blot was developed using a higher sensitivity ECL substrate.

Exposure time: 127 seconds.

 

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling Angiotensinogen with ab276132 at 1/500 (0.854 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human kidney. The section was incubated with ab276132 for 30 minutes at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

All lanes : Anti-Angiotensinogen antibody [EPR24118-2] (ab276132) at 1/1000 dilution

Lane 1 : Human plasma
Lane 2 : Rat plasma

Lysates/proteins at 20 µl per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/100000 dilution

Predicted band size: 53 kDa
Observed band size: 48-70 kDa why is the actual band size different from the predicted?

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

Angiotensinogen undergoes glycosylation. The molecular weight observed is consistent with what have been described in literature(PMID: 9694881).

Exposure times: 

Lane 1: 15 seconds
Lane 2: 3.25 seconds

 

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Angiotensinogen with ab276132 at 1/500 (0.854 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human liver. The section was incubated with ab276132 for 30 minutes at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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