Fibronectin is a high-molecular weight (~440kDa) glycoprotein found in the extracellular matrix and in blood plasma. It is made up of two subunits that vary in size between 235-270 kDa, due to alternate splicing, (Figure 1). The secreted fibronectin dimer is a soluble protein which polymerizes to higher order fibrils in the ECM. Fibronectin plays a major role in cell adhesion, growth, migration, actin dynamics and differentiation, and it is important for processes such as wound healing and embryonic development (2). Many of these functions are mediated through fibronectin binding to integrin receptor proteins (2). Altered fibronectin expression, degradation, and organization has been associated with a number of pathologies, including cancer and fibrosis (3).
In addition to integrins, fibronectin also binds extracellular matrix components such as collagen, fibrin and heparan sulfate proteoglycans (e.g. syndecans).
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Robinson, E. E., Foty, R. A. and Corbett, S. A. (2004). Fibronectin matrix assembly regulates α5β1-mediated cell cohesion. Mol. Biol. Cell 15, 973-981.
Brock, A., Chang, E., Ho, C. C., LeDuc, P., Jiang, X., Whitesides, G. M. and Ingber, D. E. (2003). Geometric determinants of directional cell motility revealed using microcontact printing. Langmuir 19, 1611-1617.
Jacob A., et al. 2013. Rab40b regulates MMP2 and MMP9 trafficking during invadopodia formation and breast cancer cell invasion. J. Cell Sci. doi: 10.1242/jcs.126573
Lively and Schlichter, 2013. The microglial activation state regulates migration and roles of matrix-dissolving enzymes for invasion. J. Neuroinflammation. 10:75.
Question 1: Can I coat cells with fluorescent ECM proteins?
Answer 1: Yes, cells can be coated with fluorescently-labeled ECM proteins such as fibronectin. Example protocols are found in Pallis et al., 1997 (Peripheral Blood Lymphocyte Binding to a Soluble FITC-Fibronectin Conjugate. Cytometry. 28, 157-164) and Huveneers et al., 2008 (Binding of soluble fibronectin to integrin a5b1 – link to focal adhesion redistribution and contractile shape. J. Cell Sci. 121, 2452-2462). Briefly, cells are incubated with a low concentration of soluble fluorescently-labeled fibronectin in an appropriate buffer. Low concentrations of fibronectin are recommended to minimize non-specific binding. The cells/fibronectin mixture is incubated in the dark for 30 min at room temperature. Others have done the incubation at 4°C for 1 hour. The cells are then rinsed in an appropriate buffer, resuspended in the same buffer, and then the coating/binding of ECM proteins can be quantified by flow cytometry or the coated cells can be used experimentally.