E Cadherin (CDH1)

Structure of the E Cadherin target protein

Figure 1: Structure of the E Cadherin target protein.

E Cadherin Target Introduction

Protein Function

Cadherins are calcium-dependent cell adhesion proteins. When connecting cells, they preferentially interact with themselves in a homophilic manner. Therefore, cadherins may contribute to the classification of different cell types.
E-Cadherin is a single-pass type I transmembrane glycoprotein located at epithelial cells' adherens junctions (AJ) and basolateral membrane. It comprises a larger extracellular domain, a transmembrane segment, and a conserved cytoplasmic domain.
An α-secretase located on the cytoplasmic membrane surface can cleave E-Cadherin. E-cadherin can be catalyzed by several proteases including matrix metalloproteinases (MMP-3, MMP-7, MMP-9, MT1-MMP), disintegrins, metalloproteinases (ADAM10, ADAM15), plasmin, and kallikrein 7, converting the mature 120 kDa E-Cadherin protein into a soluble N-terminal 80 kDa soluble E-Cadherin (sE-cad) and a C-terminal 38 kDa fragment inside the cell. sE-cad is released from the cytoplasmic membrane and diffuses into the extracellular environment, even the blood, as a paracrine/autocrine signaling molecule. In contrast, the intracellular C-terminal fragment (E-cad/CTF1) remains embedded in the cytoplasmic membrane until it is cleaved by γ-secretase (presenilin-1/2) on the intracellular surface, which leads to the disassembly of adherens junctions (AJ) and the release of the 33 kDa E-cad/CTF2 into the cytoplasm to exert signaling functions. This fragment can also be proteolyzed by caspase-3 to produce a functionally unknown 29 kDa fragment (E-cad/CTF3).
Involved in regulating adhesion, migration, and proliferation of epithelial cells.
Has effective invasive inhibition. It is also a ligand for the integrin α-E/beta-7.
E-Cad/CTF2 promotes non-amyloid degradation of Abeta precursor. It has a strong inhibitory effect on the generation of APP C99 and C83.

Protein Expression

Not expressed in neural epithelial tissue.

Protein Localization

Cell junctions, cell membrane, endosomes, Golgi apparatus.

E Cadherin ICC experimental result

Image 2: E Cadherin ICC experimental result image, Anti-E Cadherin antibody [EP700Y] - cell adhesion marker (ab40772). Green: E Cadherin, Red: Tubulin, Blue: DAPI.

Isoforms & post-translational modifications

Human (P12830): Isoforms 1-2: 91-97 kDa (predicted)
Mouse (P09803): Isoform 1: 98 kDa (predicted)
Rat (Q9R0T4): 99 kDa (predicted)
Existence of glycosylation, phosphorylation, ubiquitination modifications

WB experiment tips

Precautions

E Cadherin is a membrane protein with multiple calcium-binding protein repeat sequences in the extracellular region, which may mask antigenic epitopes. Therefore, we recommend using sonication lysate to expose antigenic epitopes for antibody binding.
In different samples, one or more bands with molecular weights of approximately 80 kDa, 97 kDa, and 120 kDa may be detected.
The expression level of E Cadherin may vary in different tissues or cells, so it is recommended to set a positive control.

Positive control

MCF-7, HT-29 cell lysate

Negative control

MDA-MB-231 cell lysate

WB- Anti-E Cadherin antibody

Figure 3: WB- Anti-E Cadherin antibody [EP700Y] - Cell adhesion Marker (ab40772).

Lane 1: 20 µg MCF-7 cell lysate
Lane 2: 20 µg HT-29 cell lysate
Lane 3: 20 µg PC-3 cell lysate
Lane 4: 20 µg MDA-MB-231 cell lysate

Primary antibody: Anti-E Cadherin antibody [EP700Y] - Cell adhesion Marker (ab40772), diluted at 1/10000 concentration.
Secondary antibody: HRP-conjugated goat anti-rabbit IgG (ab97051), diluted at 1/100000 concentration.
Predicted band size: 97 kDa

Key control points

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

Sample preparation:

Add a complex protease 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.

Antibody incubation:

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