Figure 1: Fibroblast activation protein, alpha target protein homodimer structure.

Fibroblast activation protein, alpha introduction

Protein function

• Fibroblast activation protein, alpha (FAP-alpha), is a cell-surface serine protease with dipeptidyl peptidase and endopeptidase activity, cleaving substrates at the N-terminus Xaa-Pro sequence. It participates in extracellular matrix degradation and involves many cellular processes, including tissue remodeling, fibrosis, wound healing, inflammation, and tumor growth.
• FAP-alpha is underexpressed in normal tissues but is selectively expressed in tissue remodeling and repairing, and is considered a special marker for (myo)fibroblast activation.
• FAP-alpha is highly upregulated in various cancers and promotes tumor cell migration and invasion through ECM degradation. It is often used as a marker for cancer-associated fibroblasts.
• As a membrane-anchored cell surface protein, FAP-alpha can be hydrolyzed by other proteolytic enzymes in a soluble form in tissue fluid and plasma.
• The monomeric form of FAP-alpha is inactive, and both the membrane-anchored form and the soluble form require homodimerization to be active.

Protein expression

• FAP-alpha is expressed in tissue remodeling during development, tissue repair, and fibroblasts in carcinogenic tissues.

Protein characteristics

• FAP-alpha exists in two forms, the membrane-anchored and soluble forms, after cleavage of 23 amino acids. Western blot may detect multiple bands with similar molecular weights.

Protein localization

• FAP-alpha is mainly localized to the cell membrane. It is found on the surface with lamellipodia and invadopodia membranes and on shed vesicles. It is also in the extracellular region and colocalizes with extracellular components (ECM), such as collagen fibers and fibronectin.
• The soluble form of FAP-alpha is also localized in the extracellular region, plasma, and tissue fluid.

Image 2: Fibroblast activation protein, alpha IHC image, Anti-Fibroblast activation protein, alpha antibody (ab218164)

Sample name: formalin-fixed, paraffin-embedded mouse intestine tissue.
Red: FAP-alpha, Blue: DAPI

Isoforms & Post-translation modifications

Human (Q12884): Isoform 1; 88 kDa (predicted);
                             Isoform 2; 27 kDa (predicted)

Mouse (P97321): Isoforms 1-3; 84-88 kDa (predicted)
FAP-alpha undergoes multi-site N-glycosylation modifications.
FAP-alpha can be cleaved off 23 amino acids, forming a soluble form of anti-fibrinolytic enzyme (APCE).

WB experiment tips

Precautions:

• There are two isoforms of FAP-alpha in humans, with a significant difference in molecular weight (27-88 kDa), which may result in multiple bands in WB experiments.
• FAP-alpha undergoes multiple site glycosylation modifications, so the detected bands may not match the predicted size. Please do not strip the membrane or retain the portion below 130 kDa.
• FAP-alpha expression varies in different tissues and cells, and some samples may show weak or no expression. Please choose appropriate experimental samples and use a positive control to confirm that the system works properly.

Positive control:

• HFF-1 (Human foreskin fibroblast cell line) whole cell lysate

Negative control (low or no expression):

• Human fetal liver lysate.

Example of results:

Figure 3: WB Anti-Fibroblast activation protein, alpha antibody [EPR20021] (ab207178)

Lane 1: WI-38 (human fetal lung fibroblast cell line) whole cell lysate.
Lane 2: U-87 MG (human glioblastoma-astrocytoma epithelial cell line) whole cell lysate.
Lane 3: Human fetal liver lysate (negative control).

Predicted band size: 88 kDa.
Observed band size: 95 kDa.

Figure 4: WB  Anti-Fibroblast activation protein, alpha antibody [SP325] (ab227703)

Sample name: HFF-1 (human skin fibroblast) cell lysate.
Lane 1: Incubated with Fibroblast activation protein antibody.
Lane 2: Incubated with GAPDH antibody.
Predicted band size: 88 kDa.
Observed band size: 95 kDa.

Key control points

In WB experiments, besides paying attention to general steps, it is important to focus below key control points:

Sample preparation:

1. It is recommended to boil the samples mildly.
2. Add a protease inhibitor cocktail to prevent degradation of target proteins.
3. Keep samples on ice throughout the entire sample preparation process.
4. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.

Transferring:

1. It is recommended to stain the membrane with Ponceau S after the transfer to confirm the success of the transfer.

Antibody incubation:

1. Select a suitable antibody working concentration according to the product datasheet.

IHC experiment tips

Precautions

• FAP-alpha is not expressed in normal tissues but is highly expressed in some tumor tissues. The expression varies in different tissues, so please choose appropriate experimental samples and use positive controls to confirm that the experimental system is working correctly.
• Positive staining of FAP-alpha can be observed in the extracellular matrix of tissues or on the surface of fibroblasts in the matrix.

Positive control

Human pancreatic cancer tissue.

Negative control (low or no expression)

Human normal liver tissue.

Example of results.

Figure 5: IHC- Anti-Fibroblast activation protein, alpha antibody [EPR20021] (ab207178)

Sample name: Paraffin-embedded human pancreatic cancer tissue.
Antigen retrieval method: Heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0.
Experimental results: Cell surface staining on stromal cells of human pancreas cancer is observed.

Figure 6: IHC-Anti-Fibroblast activation protein, alpha antibody [SP325] (ab227703)

Sample name: Formalin/PFA fixed, paraffin-embedded human colon cancer tissue.
Antigen retrieval method: Heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 10 mins.
Experimental results: Positive staining of the stroma in human colon cancer.

Key control points

In IHC experiments, besides paying attention to general steps, it is important to focus on the key control points:

Sample fixation:

• The optimal fixation time for samples depends on the size and type of tissue, but for most samples, it is advisable to fix with 4% PFA at room temperature for 18-24 hours.

Blocking:

• If using HRP conjugate for detection, treat the sections with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity.

Antigen retrieval:

• When performing immunohistochemistry experiments on paraffin sections, please choose the appropriate antigen retrieval solution according to the datasheet. We recommend using a pressure cooker for heat-induced antigen retrieval. You can try to repair the sections at 110℃ for 15 minutes.

Reference

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