Figure 1: Fibronectin Structure

Fibronectin Introduction

Protein Function

• Fibronectin is a multifunctional extracellular matrix protein and a high molecular weight glycoprotein. It can bind to various cell surface and extracellular matrix compounds, including collagen, fibrinogen, heparin, DNA, and actin.
• Fibronectin can be categorized as plasma fibronectin (FN) and cellular fibronectin (cFN). It presents in a soluble dimeric form in plasma and a dimeric or multimeric form at the cell surface and in the extracellular matrix. Hepatocytes secrete plasma fibronectin, circulate in a non-active, compact bloodstream conformation, and assemble into fibronectin fibrils. Cellular fibronectin is produced in various tissues by resident cells, including connective tissues, bone, and periodontal tissues.
• Fibronectin is involved in cell adhesion and migration processes, including embryogenesis, wound healing, blood clotting, fibrosis, and tumor progression.

Protein Expression

• Hepatocytes secrete fibronectin in the plasma (soluble dimeric form).
• Cellular FN (dimeric or cross-linked multimeric forms), made by fibroblasts, epithelial, and other cell types, is deposited as fibrils in the extracellular matrix.

Protein Characteristics

• Fibronectin has three alternative splicing regions that can produce 20 different transcript variants.
• Fibronectin is a high molecular weight glycoprotein and undergoes various post-translational modifications.

Protein Localization

• Fibronectin is mainly present in the extracellular matrix.

Figure 2: Fibronectin ICC experimental result image, recombinant Anti-Fibronectin antibody [EPR23110-46] (ab268020)

Sample name: NIH/3T3 (mouse embryonic fibroblast cell line)

Green: Fibronectin, Red: Tubulin, Blue: DAPI

Experimental result: Confocal image shows cytoplasmic staining in NIH/3T3 cell line.

Isoforms & Post-translation modifications

• Human (P02751):
  Isoforms 2, 16 (P02751-2, P02751-16): 72-74 kDa (predicted)
  Isoforms 4, 12 (P02751-4, P02751-12): 221-223 kDa (predicted)
  Isoforms 1, 3 (P02751-1, P02751-3): 259-263 kDa (predicted)
  Isoforms 5-11 (P02751-5-P02751-11): 240-269 kDa (predicted)
  Isoforms 13, 14, 17 (P02751-13, P02751-14, P02751-17): 249-257 kDa (predicted)
• Mouse (P11276): 273 kDa (predicted)
• Rat P04937):
  Isoforms 1-4 (P04937-1-P04937-4): 259-273 kDa (predicted)

• Fibronectin may undergo sulfation modification
• Glycosylation modification
• Phosphorylation modification
• Cross-linking

WB experiment tips

Precautions

• Fibronectin undergoes post-translational modifications such as glycosylation, and the actual detection band may not match the predicted value. A smear may appear at approximately 285 kDa on the membrane (Figure 3). We strongly recommend not cutting the membrane to avoid losing the target band.
• Fibronectin is a high-molecular-weight glycoprotein, and we recommend using conditions for electrophoretic transfer of high-molecular-weight proteins for the experiment.
• Fibronectin can be divided into plasma fibronectin (FN) and cellular fibronectin (cFN). Please select appropriate experimental samples, as some may show weak or no expression. We recommend using a positive control to confirm that the experimental system works appropriately.

Positive control

• Human serum

Example of results

Figure 3: Recombinant Anti-Fibronectin antibody [EPR23110-25] (ab268021)

Lane 1: Human serum
Lane 2: Human plasma
Lane 3: Mouse plasma
Lane 4: Rat plasma

Predicted band size: 262 kDa
Observed band size: 285 kDa

Figure 4: Anti-Fibronectin antibody [EPR23110-46] (ab268020)

Lane 1: Human serum
Lane 2: Human plasma
Lane 3: Human kidney tissue lysate
Lane 4: Human stomach tissue lysate
Lane 5: Mouse plasma
Lane 6: Rat plasma

Predicted band size: 262 kDa
Observed band size: 285 kDa

Key control points

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

Sample preparation:

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

Electrophoresis:

1. For electrophoresis, target proteins with a higher molecular weight use a lower gel percentage (6-8%).
2. Load at least 20 μg of total protein from cell lysate or tissue homogenate.

Transfer:

1. We recommend staining the membrane with Ponceau S after the transfer to confirm the transfer's success.
   It is recommended not to cut the membrane and keep the entire membrane for antibody incubation.
2. For target proteins with a higher molecular weight, we advise using a PVDF membrane with a pore size of 0.45 μm.
3. We recommend using 10% methanol or a lower concentration in the transfer buffer for target proteins with a higher molecular weight.
4. For target proteins with a higher molecular weight, it is recommended that SDS be added to the transfer buffer at a final concentration of 0.1%.

Antibody incubation:

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

Reference

1. Jennifer Patten, Karin Wang. Fibronectin in development and wound healing. Adv Drug Deliv Rev. 2021 Mar;170:353-368.doi: 10.1016/j.addr.2020.09.005. Epub 2020 Sep 19.
2. Mayra Paolillo, Sergio Schinelli. Extracellular Matrix Alterations in Metastatic Processes. Int J Mol Sci. 2019 Oct 7;20(19):4947. doi: 10.3390/ijms20194947.
3. Purva Singh, Cara Carraher, Jean E Schwarzbauer. Assembly of fibronectin extracellular matrix. Annu Rev Cell Dev Biol. 2010;26:397-419. doi: 10.1146/annurev-cellbio-100109-104020.
4. Janna K Mouw, Guanqing Ou, Valerie M Weaver. Extracellular matrix assembly: a multiscale deconstruction. Nat Rev Mol Cell Biol. 2014 Dec;15(12):771-85. doi: 10.1038/nrm3902. Epub 2014 Nov 5.