Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized rat kidney tissue labeling Angiotensin Converting Enzyme 1 with ab254222 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution (green). Positive staining on proximal tubules of kidney (PMID:10504496) is observed. The nuclear counter stain is DAPI (blue).

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

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

 

All lanes : Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (ab254222) at 1/1000 dilution

Lane 1 : HUVEC (human umbilical vein endothelial cell line) whole cell lysate at 20 µg
Lane 2 : Wild-type HAP1 whole cell lysate at 40 µg
Lane 3 : Angiotensin Converting Enzyme 1 knockout HAP1 whole cell lysate at 40 µg
Lane 4 : Human kidney cell lysate at 40 µg
Lane 5 : Human lung cell lysate at 40 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 150 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lanes 1-3: 3 minutes. Lanes 4-5: 5.5 seconds.

ab254222 was shown to specifically react with Angiotensin Converting Enzyme 1 in wild-type HAP1 cells as signal was lost in Angiotensin Converting Enzyme 1 knockout cells. Wild-type and Angiotensin Converting Enzyme 1 knockout samples were subjected to SDS-PAGE. ab254222 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique.

The molecular weight observed, and the expression profile are consistent with what have been described in the literature (PMID: 25495544, 16203874).

 

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling Angiotensin Converting Enzyme 1 with ab254222 at 1/4000 dilution, followed by a Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on proximal tubules of mouse kidney (PMID: 2828286; PMID: 175444) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

The section was incubated with ab254222 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized mouse kidney tissue labeling Angiotensin Converting Enzyme 1 with ab254222 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution (green). Positive staining on proximal tubules of kidney (PMID:10504496) is observed. The nuclear counter stain is DAPI (blue).

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

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling Angiotensin Converting Enzyme 1 with ab254222 at 1/4000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on proximal tubules in mouse kidney (PMID: 2828286; PMID: 175444) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

The section was incubated with ab254222 for 30 mins at room temperature. 
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

All lanes : Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (ab254222) at 1/1000 dilution

Lane 1 : Rat brain tissue lysate
Lane 2 : Rat heart tissue lysate
Lane 3 : Rat liver tissue lysate
Lane 4 : Rat spleen tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 150 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lane 1: 48 seconds. Lanes 2 & 3:  3 minutes. Lane 4: 48 seconds.

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling Angiotensin Converting Enzyme 1 with ab254222 at 1/4000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on blood vessels of human liver (PMID: 175444) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

The section was incubated with ab254222 for 30 mins at room temperature. 
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of paraffin-embedded human lkidney tissue labeling Angiotensin Converting Enzyme 1 with ab254222 at 1/4000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on proximal tubules of human kidney (PMID: 2828286; PMID: 175444) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

The section was incubated with ab254222 for 30 mins at room temperature. 
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

All lanes : Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (ab254222) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse heart tissue lysate
Lane 3 : Mouse kidney tissue lysate
Lane 4 : Mouse spleen tissue lysate
Lane 5 : Mouse lung tissue lysate
Lane 6 : bEND.3 (mouse brain endothelioma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 150 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lanes 1-4: 10 seconds. Lane 5: 5.5 seconds. Lane 6: 48 seconds.

ELISA analysis of ACE recombinant protein at 1000 ng/mL with ab254222. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.