Fibronectin Coating Protocol

Important points to remember before you start

1. Ensure the cells are healthy and in adequate numbers.
2. Fibronectin products provided as solutions can be stored for at least a year at 2 - 8 °C if kept sterile.
3. Reconstitute fibronectin products provided as lyophilized powders in suitable solvents. Dissolve for at least 30 minutes at 37 °C. A small amount of undissolved material may remain. This will not affect product performance. Reconstituted fibronectin solution can be stored in working aliquots at -20 °C or lower.
4. Vortexing or excessive agitation of fibronectin solutions should be avoided.
5. Fibronectin coated cultureware can be stored for 2-4 weeks at 2-8 °C in a closed sterile container or sterile sealable bags.
6. Surface coverage concentration may differ with the cells being cultured. Protocol optimization is recommended to determine actual coating concentration for your cells. Recommended concentrations are listed in Table 1.
7. Do not over-dry culture plates/coverslips post coating as this affects the attachment of cells.
8. All the steps in the protocols need to be performed in sterile conditions.

Fibronectin coating protocol for culture ware

1. Dilute fibronectin to the desired concentration. Optimum conditions for attachment are dependent on cell type and application. The typical coating concentration is 1 – 5 ug/cm².
2. Coat culture surface with a minimal volume.
3. Once the entire area is covered, remove excess solution. This can be used to coat another well/plate.
4. Air dry at least for 45 minutes at room temperature. before introducing cells and medium.

Table 1.Fibronectins for cell attachment

Product No.	Description	Coating concentration	Dilute in
10838039001	Fibronectin from human plasma	5 μg/cm2	Sterile water
FIBRP-RO	Fibronectin (pure)	5 μg/cm2	Sterile water
F0556	Fibronectin solution human fibroblasts	1-5 μg/cm2	sterile HBSS
F0635	Fibronectin rat plasma	1-5 μg/cm2	Sterile water
F0895	Fibronectin human plasma	1-5 μg/cm2	Sterile balanced salt solution
F1056	Fibronectin human plasma	1-5 μg/cm2	Sterile water
F1141	Fibronectin bovine plasma	1-5 μg/cm2	Sterile water
F2006	Fibronectin human plasma	1-5 μg/cm2	Sterile water
F2518	Fibronectin human foreskin fibroblasts	1-5 μg/cm2	Sterile water
F4759	Fibronectin bovine plasma	1-5 μg/cm2	Sterile water
FC010	Human Plasma Fibronectin Purified Protein	2-10 μg/cm2	Sterile balanced salt solution
FC014	Fibronectin, Bovine	2-10 μg/cm2	sterile balanced salt solution
S5171	Superfibronectin from human plasma	1 μg/cm2	Sterile PBS
ECM001	Fibronectin human, recombinant, expressed in HEK	1-5 μg/cm2	Sterile water

Fibronectin Proteolytic Fragments

Fibronectin fragments are obtained from plasma fibronectin by using proteolytic enzymes. These fragments are useful tools for mapping regions within the fibronectin molecule, which are responsible for specific binding to collagen and for other biologically relevant functions.

Table 2.Fibronectin fragments for binding studies

Product No.	Description	Binding activity
F9911	Fibronectin Proteolytic Fragment from human plasma, 30kDa	Heparin-binding fragment
F0162	Fibronectin Proteolytic Fragment from human plasma, 45kDa	Gelatin-binding fragment
F0287	Fibronectin Proteolytic Fragment from human plasma, 70kDa	Heparin- and gelatin-binding fragment
F3542	Fibronectin Fragment III1-C human	Fibronectin-binding fragment (self-assembly)
F1903	Fibronectin 40 kDa α Chymotryptic Fragment (Heparin-binding region), Human purified	Heparin-binding fragment
F1904	Fibronectin 120 kDa α Chymotryptic Fragment (Cell Attachment region), Human purified	cell attachment region of fibronectin

Frequently Asked Questions (FAQs)

1. What is the purity of fibronectin products?

We do not determine the purity of fibronectin solutions. An SDS PAGE gel is run and one major fibronectin band of 120kDa is compared to past lots to ensure consistent quality. All our fibronectin powders are ≥95% pure.

2. What is the best solvent to dilute fibronectin?

The solvents that are compatible to dilute fibronectins are provided in Table 1

3. How long can fibronectin be stored?

Fibronectin products provided as solutions can be stored for a year at 2 - 8 °C provided they are sterile. Diluted, unused fibronectin can be kept at 2 - 8 °C for up to 1 week. Fibronectin-coated plates can be stored at 2 - 8 °C for up to 4 weeks provided they are sealed well to prevent contamination and/or drying up of fibronectin. Do not use the product if discoloration or cracks appear on the surface of the coated material

4. What is the optimum concentration of fibronectin for coating?

The coating concentrations of our fibronectins are suggested in Table 1. However, optimizing the concentrations that work for your cells is recommended.

5. What is the best ECM protein to study stem cell differentiation?

While laminin and fibronectin are widely used ECM proteins, the one that your cell needs should be determined by test experiments. Stem cells express varied integrins on the surface during different stages of differentiation. The choice of ECM protein would depend on the stage of differentiation you are interested in studying.