All lanes : Anti-ACE2 antibody [EPR4436] (ab108209) at 1/1000 dilution

Lane 1 : Wild-type HepG2 cell lysate
Lane 2 : ACE2 knockout HepG2 cell lysate
Lane 3 : Calu-3 cell lysate
Lane 4 : A549 cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Predicted band size: 92 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab108209 observed at 130 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab108209 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 ab108209 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 labelling ACE2 with ab108209 at 1/100. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

All lanes : Anti-ACE2 antibody [EPR4436] (ab108209) at 1/1000 dilution

Lane 1 : Wild-type Caco-2 cell lysate
Lane 2 : ACE2 knockout Caco-2 cell lysate
Lane 3 : Calu-3 cell lysate
Lane 4 : A549 cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Predicted band size: 92 kDa
Observed band size: 125 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab108209 observed at 125 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab108209 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 ab108209 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.

All lanes : Anti-ACE2 antibody [EPR4436] (ab108209) at 1/1000 dilution

Lane 1 : Human testis cell lysate
Lane 2 : Human kidney cell lysate
Lane 3 : Human lung cell lysate
Lane 4 : HepG2 cell lysate
Lane 5 : Caco-2 cell lysate
Lane 6 : A549 cell lysate (negative control)

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 92 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

Lanes 1 - 6: Merged signal (red and green). Green - ab108209 observed at 120 kDa. Red - loading control, Mouse anti-Actin observed at 42kDa.

ab108209 was shown to react with ACE2 in western blot. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab108209 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).

All lanes : Anti-ACE2 antibody [EPR4436] (ab108209)

Lane 1 : Human fetal kidney lysate
Lane 2 : Human testis lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 92 kDa
Observed band size: 115 kDa why is the actual band size different from the predicted?

ab108209 Immunoprecipitating ACE2 in human testis tissue lysate. 0.35 mg of tissue lysate was incubated with 2 μg primary antibody (1/30). 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.

All lanes : Anti-ACE2 antibody [EPR4436] (ab108209) at 1/500 dilution

Lane 1 : Human testis tissue lysate at 10 µg
Lane 2 : ab108209 + Human testis tissue lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab108209 in Human testis tissue lysate

Observed band size: 110 kDa why is the actual band size different from the predicted?

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue labelling ACE2 with ab108209 at 1/100. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.