Recombinant Anti-ACE2 antibody [EPR4435(2)] (ab108252)

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

ab181602 was used as a GAPDH loading control.

Two bands observed by ab108252 corresponding to glycosylation and non-glycosylation forms.

Signal in heart tissue is low, we recommend loading more amount of lysate or using lower antibody dilution to improve result.

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

ab181602 was used as GAPDH loading control.

Exposure time: Lane 1: 7 seconds; Lane 2-8: 180 seconds.

Two bands observed by ab108252 corresponding to glycosylation and non-glycosylation forms.

Signal in mouse and rat tissues are low, we recommend loading more amount of lysate or using lower antibody dilution to improve result.

Lanes 1 - 4: Merged signal (red and green). Green - ab108252 observed at 130 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab108252 was shown to react with ACE2 in wild-type HepG2 cells in western blot with loss of signal observed in ACE2 knockout cell line ab273733 (knockout cell lysate ab275495). Wild-type and ACE2 knockout HepG2 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab108252 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labeling ACE2 with ab108252 at 1/6400 dilution. Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. Staining was visualised using Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

The section was incubated with ab108252 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Lanes 1 - 4: Merged signal (red and green). Green - ab108252 observed at 125 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab108252 was shown to react with ACE2 in Caco-2 wild-type cells in western blot with loss of signal observed in ACE2 knockout cell line ab273731 (knockout cell lysate ab275516). Wild-type and ACE2 knockout Caco-2 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab108252 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Lanes 1 - 6: Merged signal (red and green). Green - ab108252 observed at 120 kDa. Red - loading control, Mouse anti-Actin observed at 42kDa.

ab108252 was shown to react with ACE2 in western blot. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab108252 and Mouse anti Actin overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Absence of ACE2 expression in A549 cells aligns with previously reported mRNA and protein data (PMID 16282461; fig.2b and 2c).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue labeling ACE2 with ab108252 at 1/6400 dilution. Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. Staining was visualised using Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

The section was incubated with ab108252 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue labeling ACE2 with ab108252 at 1/6400 dilution. Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. Staining was visualised using Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

The section was incubated with ab108252 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

ab108252 Immunoprecipitating ACE2 in human testis tissue lysate. 0.35 mg of tissue lysate was incubated with 0.6 μg primary antibody (1/20). For western blotting a HRP-conjugated Veriblot for IP Detection Reagent (ab131366) (1/1000) was used to confirm successful immunoprecipitation.

Exposure time: 1 second.
Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.

ELISA using ab108252 at varying antibody concentrations (4000~0 ng/ml) and antigen concentration at 1000 ng/mL. An Alkaline Phosphatase-conjugated Goat Anti-Rabbit IgG (H+L) (1/2500) was used as the secondary antibody.