N-cadherin (CDH2)

Structure of the N-cadherin target protein

Figure 1: Structure of the N-cadherin target protein.

Introduction to the N-cadherin

Protein Function

N-cadherin, also known as Neural-cadherin (Cadherin-2), is a calcium-dependent single-chain transmembrane glycoprotein mediates homotypic and heterotypic cell adhesion. N-cadherin comprises a hydrophobic transmembrane region, an extracellular domain, and a highly conserved C-terminal intracellular domain.
The cadherin family consists of intercellular adhesion molecules primarily distributed in various epithelial tissues, mediating cell-cell adhesion. The classic cadherin family includes N-, P-, R-, B-, and E-cadherins.
N-cadherin is a vital member of the cadherin family, playing a crucial role in the development and functional regulation of the nervous system, brain, heart, skeletal muscle, blood vessels, and hematopoietic microenvironment.
Abnormal expression of N-cadherin is closely related to the progression of human malignancies, including transformation, adhesion, apoptosis, angiogenesis, invasion, and metastasis.

Protein Expression

N-cadherin is widely expressed in embryos, participating in developing and regulating neural tissues, the brain, the heart, and other organs.
In mature mammals, N-cadherin is only distributed in neural tissues, fibroblasts, and mesodermal cells.

Protein Localization

N-cadherin is primarily located in the cell membrane and cell junctions.

N-cadherin ICC Experimental Results, Anti-N Cadherin Antibody [8C11] (ab19348)

Figure 2: N-cadherin ICC Experimental Results, Anti-N Cadherin Antibody [8C11] (ab19348)

Sample Name: SH-SY5Y (human bone marrow neuroblastoma cell line).

Experimental Conditions: Fixed with 100% methanol for 5 minutes, permeabilized with 0.1% Tween for 5 minutes, then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour.

Experimental Results: Green: N-cadherin ; Red: Tubulin; Blue: DAPI

Isoforms & Post-translational Modifications

Human (P19022): Isoform 1: 99 kDa; Isoform 2: 97 kDa (predicted)
Mouse (P15116): 100 kDa (predicted)
Rat (Q9Z1Y3): 100 kDa (predicted)
N-cadherin undergoes glycosylation modifications.
N-cadherin undergoes phosphorylation modifications.
N-cadherin can potentially be cleaved by MMP24. Cleavage of the extracellular domain results in a 90 kDa N-terminal soluble fragment and a 45 kDa membrane-bound C-terminal fragment 1 (CTF1).

WB Experiment Tips

Precautions

Due to glycosylation modifications of N-cadherin, protein band migration may be affected. This could result in multiple bands being detected between 110-140 kDa, which may not match the predicted value of 99 kDa (as shown in Figure 3).
We recommend retaining the entire membrane to detect N-cadherin and prevent the loss of the target band. Expression of N-cadherin varies across different tissues and cells; some samples may exhibit weak or no expression (e.g., MCF7). We recommend using experimentally validated samples from the literature. We strongly recommend including positive controls (e.g., mouse/rat brain tissue and HepG2) to properly ensure the experimental system functions.
N-cadherin may undergo cleavage, and some samples may show a 45 kDa fragment band.
We recommend using freshly prepared lysis buffer to detect N-cadherin and minimize protein degradation.

Positive Controls

HepG2 cell line
Mouse brain tissue
Rat brain tissue

Negative Control (Low or No Expression)

MCF7 cell line

Example Results

Recombinant Anti-N Cadherin Antibody [EPR1791-4] (ab76011)

Figure 3: Recombinant Anti-N Cadherin Antibody [EPR1791-4] (ab76011)

Lane 1: HeLa cell lysate
Lane 2: HeLa cell lysate (scraped collection)
Lane 3: Human brain tissue lysate
Lane 4: Mouse brain tissue lysate
Lane 5: Rat brain tissue lysate
Lane 6: MCF7 cell lysate (negative control)

Predicted Band Size: 100 kDa
Observed Band Size: 125 kDa

Experimental Results: Green: N-cadherin; Red: GAPDH

Recombinant Anti-N Cadherin Antibody [32/N-Cadherin] (ab280375)

Figure 4: Recombinant Anti-N Cadherin Antibody [32/N-Cadherin] (ab280375)

Lane 1: Mouse brain tissue lysate
Lane 2: Mouse kidney tissue lysate
Lane 3: Rat heart tissue lysate
Lane 4: C6 cell lysate
Lane 5: PC-12 cell lysate

Predicted Band Size: 99 kDa
Observed Band Size: 140 kDa

Key control points

In addition to the routine issues that need to be paid attention to in the experiment, special attention should be paid to the following critical control points:

Sample preparation:

Use freshly prepared lysis buffer.
Use a combination of protease inhibitors to prevent the target protein from being degraded.
Keep the sample on ice during the entire sample preparation process.
Determine the total protein concentration of the sample by Bradford analysis, Lowry analysis, or BCA analysis.
Set up positive and negative controls.

Electrophoresis:

Load at least 20 μg of total protein for electrophoresis.

Transfer:

It is strongly recommended to use Ponceau red staining after transfer to determine whether the transfer is successful. We also recommend not cutting the membrane.
For target proteins with larger molecular weights, 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 a 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 datasheet.