Angiotensin-converting enzyme 2 (ACE2)

ACE2 target protein structure

Figure 1: ACE2 target protein structure.

ACE2 Target Introduction

Protein Function

ACE2, also known as angiotensin-converting enzyme II, is an important member of the renin-angiotensin system (RAS).
As a carboxypeptidase, ACE2 can convert angiotensin I to angiotensin 1-9, or convert angiotensin II to angiotensin 1-7. It is a key regulator of blood volume, systemic vascular resistance, and cardiovascular homeostasis.
ACE2 is also a type I integral membrane protein, mainly located in the extracellular space, with its carboxyl terminus connected to the membrane through a transmembrane domain.
In order to exert its regulatory role in the RAS, ACE2 (cACE2) needs to be transported to the cell surface, where it is cleaved by host proteinases such as ADAM17, releasing the enzymatically active soluble ACE2 (sACE2) into the plasma.
ACE2 has also been found to be a receptor for SARS-CoV, SARS-CoV-2, and NL63/HCoV-NL63 coronaviruses.

Protein Expression

ACE2 is widely distributed in tissues such as the lungs, heart, kidneys, gastrointestinal system, and testes.
ACE2 is upregulated in the failing human heart.

Protein Localization

ACE2 can be localized to the cell membrane and cytoplasm.
Cleaved ACE2 is a secreted protein.

ACE2 ICC experimental results, recombinant Anti-ACE2 antibody [EPR24705-45] (ab272500)

Figure 2: ACE2 ICC experimental results, recombinant Anti-ACE2 antibody [EPR24705-45] (ab272500)

Sample name: Caco2 cell line.

Experimental conditions: Cells fixed with 4% paraformaldehyde (10 minutes), permeabilized with 0.1% Tween (5 minutes), blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween solution for 1 hour.

Experimental results: Green: ACE2, Red: Tubulin, Blue: DAPI. The top image shows WT Caco2 cell line, and the bottom image shows ACE2 knockout Caco2 cell line.

ACE2 ICC experimental result image, recombinant Anti-ACE2 antibody [EPR24705-45] (ab272500)

Figure 3: ACE2 ICC experimental result image, recombinant Anti-ACE2 antibody [EPR24705-45] (ab272500)

Sample name: Calu-3 cell line.

Experimental conditions: Cells fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100.

Experimental results: Green: ACE2, Red: Tubulin, Blue: DAPI. Confocal image shows ACE2 membrane staining in Calu-3 cell line.

Negative control: HeLa (consistent with results in the literature PMID: 32926098).

Isoforms & Post-translation modifications

Human (Q9BYF1): Isoform 1: 53 kDa; Isoform 2: 92 kDa (predicted)
Mouse (Q8R0I0): Isoform 1: 40 kDa; Isoform 2: 92 kDa (predicted)
Rat (Q5EGZ1): 92 kDa (predicted)
ACE2 undergoes glycosylation modification, N-glycosylation modification may affect the infectivity of SARS.
ACE2 undergoes phosphorylation modification.
ACE2 is cleaved by ADAM17 to produce a secreted form.
ACE2 can also be cleaved by TMPRSS2, TMPRSS11D, and HPN/TMPRSS1.

WB experiment tips

Precautions

Due to the glycosylation modification of multiple sites in ACE2, it may affect the migration of protein bands, resulting in fuzzy trailing bands (as shown in Figure 5). It is recommended to retain the whole membrane to prevent the loss of target bands.
ACE2 is expressed differently in different tissue cells, and some samples may show weak or no expression (such as HeLa cells and human liver tissue). It is recommended to use experiment samples that have been validated in the literature.Please select appropriate experimental samples and strongly recommend adding positive controls (such as
human kidney tissue, human testis tissue, and Calu-3) to confirm that the experimental system is working properly.
ACE2 has multiple post-translational modifications, which may result in band sizes detected in the experiment ranging from 120-135 kDa, which does not match the predicted value of 92 kDa.

Positive controls

Human kidney tissue
Human testis tissue
Calu-3

Negative controls (low or no expression)

HeLa cell line
Human liver tissue

Example of results

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

Figure 4: Recombinant Anti-ACE2 antibody [EPR4435(2)] (ab108252)

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 (negative control)

Predicted band size: 92 kDa
Actual detected band size: 130 kDa

Experimental results: Green: ACE2, Red: GAPDH. ACE2 signal disappears in the knockout cell line. Loss of ACE2 expression in A549 cells is consistent with the data reported in PMID: 16282461.

Recombinant Anti-ACE2 antibody [EPR24705-45] (ab272500)

Figure 5: Recombinant Anti-ACE2 antibody [EPR24705-45] (ab272500)

Lane 1: Human kidney tissue lysate
Lane 2: Human testis tissue lysate
Lane 3: Human liver tissue lysate (negative control)

Predicted band size: 92 kDa
Observed band size: 120 kDa
*Observed expression pattern/molecular weight is consistent with the description in the literature

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

Add a complex proteinase inhibitor to avoid degradation of the target protein.
Keep the sample on ice throughout the sample preparation process.
Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

Load at least 20 μg total protein for electrophoresis.

Transfer:

We recommend using Ponceau S staining after transfer to confirm the success of the transfer.
For target proteins with larger molecular weight, it is recommended to use a 0.45 μm PVDF membrane.
For target proteins with larger molecular weight, it is recommended to use 10% methanol or lower concentration in the transfer buffer.
For target proteins with larger molecular weight, it is recommended to add SDS to a final concentration of 0.1% in the transfer buffer.

Antibody incubation:

Please select the appropriate antibody working concentration according to the product manual.