Rabbit Recombinant Monoclonal Angiotensin Converting Enzyme 1 antibody. Carrier free. Suitable for mIHC, I-ELISA, WB, IHC-Fr, IHC-P and reacts with Human, Mouse, Rat, Recombinant fragment samples. Cited in 1 publication.
IgG
Rabbit
pH: 7.2 - 7.4
Constituents: PBS
Liquid
Monoclonal
mIHC | Flow Cyt | IP | ICC/IF | I-ELISA | WB | IHC-Fr | IHC-P | |
---|---|---|---|---|---|---|---|---|
Human | Tested | Not recommended | Not recommended | Not recommended | Expected | Tested | Expected | Tested |
Mouse | Expected | Not recommended | Not recommended | Not recommended | Expected | Tested | Tested | Tested |
Rat | Expected | Not recommended | Not recommended | Not recommended | Expected | Tested | Tested | Tested |
Recombinant fragment | Not recommended | Not recommended | Not recommended | Not recommended | Tested | Not recommended | Not recommended | Not recommended |
Species | Dilution info | Notes |
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Species Human | Dilution info - | Notes - |
Species | Dilution info | Notes |
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Species Mouse, Rat | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
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Species Recombinant fragment | Dilution info - | Notes - |
Species | Dilution info | Notes |
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Species Human, Mouse, Rat, Recombinant fragment | Dilution info - | Notes - |
Species | Dilution info | Notes |
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Species Human, Mouse, Rat, Recombinant fragment | Dilution info - | Notes - |
Species | Dilution info | Notes |
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Species Human, Mouse, Rat, Recombinant fragment | Dilution info - | Notes - |
Species | Dilution info | Notes |
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Species Recombinant fragment | Dilution info - | Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Species | Dilution info | Notes |
---|---|---|
Species Human, Mouse, Rat | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info - | Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Species Rat | Dilution info - | Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Species Human | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Recombinant fragment | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info - | Notes Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). |
Species Rat | Dilution info - | Notes Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Recombinant fragment | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info - | Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Species Rat | Dilution info - | Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Species Human | Dilution info - | Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Species | Dilution info | Notes |
---|---|---|
Species Recombinant fragment | Dilution info - | Notes - |
Dipeptidyl carboxypeptidase that removes dipeptides from the C-terminus of a variety of circulating hormones, such as angiotensin I, bradykinin or enkephalins, thereby playing a key role in the regulation of blood pressure, electrolyte homeostasis or synaptic plasticity (PubMed:15615692, PubMed:20826823, PubMed:2558109, PubMed:4322742, PubMed:7523412, PubMed:7683654). Composed of two similar catalytic domains, each possessing a functional active site, with different selectivity for substrates (PubMed:10913258, PubMed:1320019, PubMed:1851160, PubMed:19773553, PubMed:7683654, PubMed:7876104). Plays a major role in the angiotensin-renin system that regulates blood pressure and sodium retention by the kidney by converting angiotensin I to angiotensin II, resulting in an increase of the vasoconstrictor activity of angiotensin (PubMed:11432860, PubMed:1851160, PubMed:19773553, PubMed:23056909, PubMed:4322742). Also able to inactivate bradykinin, a potent vasodilator, and therefore enhance the blood pressure response (PubMed:15615692, PubMed:2558109, PubMed:4322742, PubMed:6055465, PubMed:6270633, PubMed:7683654). Acts as a regulator of synaptic transmission by mediating cleavage of neuropeptide hormones, such as substance P, neurotensin or enkephalins (PubMed:15615692, PubMed:6208535, PubMed:6270633, PubMed:656131). Catalyzes degradation of different enkephalin neuropeptides (Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg-Phe and possibly Met-enkephalin-Arg-Gly-Leu) (PubMed:2982830, PubMed:6270633, PubMed:656131). Acts as a regulator of synaptic plasticity in the nucleus accumbens of the brain by mediating cleavage of Met-enkephalin-Arg-Phe, a strong ligand of Mu-type opioid receptor OPRM1, into Met-enkephalin (By similarity). Met-enkephalin-Arg-Phe cleavage by ACE decreases activation of OPRM1, leading to long-term synaptic potentiation of glutamate release (By similarity). Also acts as a regulator of hematopoietic stem cell differentiation by mediating degradation of hemoregulatory peptide N-acetyl-SDKP (AcSDKP) (PubMed:26403559, PubMed:7876104, PubMed:8257427, PubMed:8609242). Acts as a regulator of cannabinoid signaling pathway by mediating degradation of hemopressin, an antagonist peptide of the cannabinoid receptor CNR1 (PubMed:18077343). Involved in amyloid-beta metabolism by catalyzing degradation of Amyloid-beta protein 40 and Amyloid-beta protein 42 peptides, thereby preventing plaque formation (PubMed:11604391, PubMed:16154999, PubMed:19773553). Catalyzes cleavage of cholecystokinin (maturation of Cholecystokinin-8 and Cholecystokinin-5) and Gonadoliberin-1 (both maturation and degradation) hormones (PubMed:10336644, PubMed:2983326, PubMed:7683654, PubMed:9371719). Degradation of hemoregulatory peptide N-acetyl-SDKP (AcSDKP) and amyloid-beta proteins is mediated by the N-terminal catalytic domain, while angiotensin I and cholecystokinin cleavage is mediated by the C-terminal catalytic region (PubMed:10336644, PubMed:19773553, PubMed:7876104).Angiotensin-converting enzyme, soluble formSoluble form that is released in blood plasma and other body fluids following proteolytic cleavage in the juxtamembrane stalk region.Isoform Testis-specificIsoform produced by alternative promoter usage that is specifically expressed in spermatocytes and adult testis, and which is required for male fertility (PubMed:1651327, PubMed:1668266). In contrast to somatic isoforms, only contains one catalytic domain (PubMed:1651327, PubMed:1668266). Acts as a dipeptidyl carboxypeptidase that removes dipeptides from the C-terminus of substrates (PubMed:1668266, PubMed:24297181). The identity of substrates that are needed for male fertility is unknown (By similarity). May also have a glycosidase activity which releases GPI-anchored proteins from the membrane by cleaving the mannose linkage in the GPI moiety. The GPIase activity was reported to be essential for the egg-binding ability of the sperm (By similarity). This activity is however unclear and has been challenged by other groups, suggesting that it may be indirect (By similarity).
DCP, DCP1, DCP1, DCP, ACE, Angiotensin-converting enzyme, ACE, Dipeptidyl carboxypeptidase I, Kininase II
Rabbit Recombinant Monoclonal Angiotensin Converting Enzyme 1 antibody. Carrier free. Suitable for mIHC, I-ELISA, WB, IHC-Fr, IHC-P and reacts with Human, Mouse, Rat, Recombinant fragment samples. Cited in 1 publication.
IgG
Rabbit
pH: 7.2 - 7.4
Constituents: PBS
Liquid
Monoclonal
Yes
EPR22291-247
Affinity purification Protein A
Blue Ice
+4°C
+4°C
Do Not Freeze
ab254278 is the carrier-free version of Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222.
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
This product is a recombinant monoclonal antibody, which offers several advantages including:
For more information, read more on recombinant antibodies.
Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.
This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.
Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with 1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.
This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.
This supplementary information is collated from multiple sources and compiled automatically.
Angiotensin Converting Enzyme 1 also known as ACE1 or ACE is an important enzyme in the renin-angiotensin system. This enzyme exhibits a significant role in blood pressure regulation. ACE1 is a zinc-metallopeptidase with a molecular weight of approximately 130 kDa. It converts angiotensin I into the potent vasoconstrictor angiotensin II a critical function in cardiovascular physiology. ACE1 is widely expressed in endothelial cells particularly in the lungs kidneys and the small intestine.
The enzyme generates angiotensin II by cleaving angiotensin I. Angiotensin II an important effector peptide impacts cardiovascular and renal systems influencing vasoconstriction and fluid balance. While not directly forming a complex ACE1's activity increases the potency of angiotensin II which binds to angiotensin II receptors to exert its effects therefore indirectly forming a functional signaling complex.
ACE1 plays a central role in the renin-angiotensin system and the kallikrein-kinin system. The enzyme's activity boosts angiotensin II production which connects it to the regulation of blood pressure via the renin-angiotensin pathway. ACE1 also indirectly interacts with proteins like bradykinin by degrading them modulating kinin-related functions and influencing inflammation and tension in vascular smooth muscle.
Understanding ACE1 is important for addressing hypertension and congestive heart failure. ACE1's conversion of angiotensin I to angiotensin II means overactivity can cause elevated blood pressure leading to hypertension. This makes ACE inhibitors such as lisinopril and ramipril therapeutic for these conditions. Furthermore its connection with aldosterone production places ACE1 in relevance to heart failure as excessive aldosterone can cause detrimental remodeling of cardiac tissue.
We have tested this species and application combination and it works. It is covered by our product promise.
We have not tested this specific species and application combination in-house, but expect it will work. It is covered by our product promise.
This species and application combination has not been tested, but we predict it will work based on strong homology. However, this combination is not covered by our product promise.
We do not recommend this combination. It is not covered by our product promise.
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In the unlikely event of one of our products not working as expected, you are covered by our product promise.
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Terms & Conditions.
Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized rat kidney tissue labeling Angiotensin Converting Enzyme 1 with Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (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) (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).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222).
Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling Angiotensin Converting Enzyme 1 with Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222 at 1/4000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (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 (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 Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222).
Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized mouse kidney tissue labeling Angiotensin Converting Enzyme 1 with Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/1000 dilution.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222).
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 Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222 at 1/4000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (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 (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 Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222).
Immunohistochemical analysis of paraffin-embedded human liver tissue labeling Angiotensin Converting Enzyme 1 with Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222 at 1/4000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (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 (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 Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222).
Immunohistochemical analysis of paraffin-embedded human lkidney tissue labeling Angiotensin Converting Enzyme 1 with Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222 at 1/4000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (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 (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 Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222).
Blocking and dilution buffer: 5% NFDM/TBST.
Exposure times.
Lanes 1-3: 3 minutes. Lanes 4-5: 5.5 seconds.
Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] 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. Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222 and Anti-GAPDH antibody [EPR16891] - Loading Control 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 (Goat Anti-Rabbit IgG H&L (HRP) 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).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222).
All lanes: Western blot - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] - BSA and Azide free (ab254278) 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
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/10000 dilution
Predicted band size: 150 kDa
This data was developed using Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222, the same antibody clone in a different buffer formulation.ELISA analysis of ACE recombinant protein at 1000 ng/mL with Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.
Fluorescence multiplex immunohistochemical analysis of the human kidney (Formalin/PFA-fixed paraffin-embedded sections).
Panel A: merged staining of anti-Hexokinase 1 (Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker ab150423, green; Opal™690), anti-Angiotensin Converting Enzyme 1 (Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222, gray; Opal™520) and anti-Aquaporin 2 (Anti-Aquaporin 2 antibody [EPR21080] ab199975, red; Opal™570) on human kidney. Panel B: anti-Aquaporin 2 stained on collecting tubules. Panel C: anti-Angiotensin Converting Enzyme 1 stained on proximal tubules. Panel D: anti-Hexokinase 1 stained on distal tubules and collecting tubules. Opal Polymer HRP Ms + Rb was used as a secondary antibody.
The section was incubated in three rounds of staining: in the order of Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker ab150423 at 1/250 dilution (4.224 μg/ml), Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222 at 1/4000 dilution (0.141 μg/ml) and Anti-Aquaporin 2 antibody [EPR21080] ab199975 at 1/4000 dilution (0.152 μg/ml) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222).
False colour image of Western blot: Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222 was shown to bind specifically to Angiotensin Converting Enzyme 1. A band was observed at 200 kDa in wild-type SKNFI cell lysates with no signal observed at this size in Ace knockout cell line ab288707. To generate this image, wild-type and Ace knockout SKNFI cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
Lanes 1 - 4: Western blot - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222) at 1/1000 dilution
Lane 1: Wild-type SKNFI cell lysate at 20 µg
Lane 2: Ace knockout SKNFI cell lysate at 20 µg
Lane 3: Human Lung cell lysate at 20 µg
Lane 4: HUVEC cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 150 kDa
Observed band size: 200 kDa
Fluorescence multiplex immunohistochemical analysis of paraffin-embedded Human kidney tissue.
Panel A: Merged staining of anti-Hexokinase 1 (gray; Opal™690), anti-Angiotensin Converting Enzyme 1 (green; Opal™520) and anti-Tissue Factor (red; Opal™570) on human kidney.
Panel B: Anti-Tissue Factor stained on renal glomeruli.
Panel C: Anti-Angiotensin Converting Enzyme 1 stained on proximal tubules.
Panel D: Anti-Hexokinase 1 stained on distal tubules.
The section was incubated in three rounds of staining: in the order of Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker ab150423, Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] ab254222, and Anti-Tissue Factor antibody [EPR22548-240] ab252918 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. Counterstained with DAPI.
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