Product Uses Include
Fibronectin purified from bovine plasma and has been modified to contain a covalently linked rhodamine fluorescent dye. An activated ester of rhodamine has been used to label the protein with a labeling stoichiometry of apprximately 1.0 dyes per protein molecule, a low labeling stiochiometry to retain functional activity. No free dye is apparent in the final product. Fibronectin has an approximate molecular weight of 250 kDa. FNR01 (20 µg of protein) is provided as a lyophilized powder.
Fluorescent Fibronectin Treated MCF10A cells
Fluorescent fibronectin (Cat. # FNR01) treated MCF10Acells (image kindly provided by A. Varadara and M. Karthykenyan, Univ. S.Carolina,Columbia, SC).
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are >90% pure. No free dye is apparent in the final product.
Figure 1: Rhodamine Fibronectin Purity Determination. A 20 µg sample of rhodamine fibronectin (molecular weight approx. 250 kDa) was separated by electrophoresis oin a 4-20% SDS-PAGE system and stained with Coomassie Blue.
Comelles J. et al. 2014. Cells as active particles in asymmetric potentials: motility under external gradients. Biophys. J. 107, 1513-1522.
Steele et al., 2012. Tandem zyxin LIM sequences do not enhance force sensitive accumulation. Biochem. Biophys. Res. Commun. 422, 653–657.
Nakayama et al., 2012. Thermoresponsive Poly(N-isopropylacrylamide)-Based Block Copolymer Coating for Optimizing Cell Sheet Fabrication. Macromol. Biosci. 12, 751–760.
Tamura et al., 2012. Thermally responsive microcarriers with optimal poly(N-isopropylacrylamide) grafted density for facilitating cell adhesion/detachment in suspension culture. Acta Biomaterialia. doi:http://dx.doi.org/10.1016/j.actbio.2012.07.006.
Steward et al., 2011. Mechanical stretch and shear flow induced reorganization and recruitment of fibronectin in fibroblasts. Sci. Rep. 1, 147. doi: 10.1038/srep00147.
Nagase et al., 2011. Thermo-Responsive Polymer Brushes as Intelligent Biointerfaces: Preparation via ATRP and Characterization. Macromol Biosci. 11, 300-309.
Robinson et al., 2004. Fibronectin matrix assembly regulates α5β1-mediated cell cohesion. Mol. Biol. Cell. 15, 973-981.
Brock et al., 2003. Geometric determinants of directional cell motility revealed using microcontact printing. Langmuir. 19, 1611-1617.
Question 1: What is the optimal excitation and emission filter settings to visualize the rhodamine fluorescence?
Answer 1: Rhodamine fibronectin can be detected using a filter set of 535 nm excitation and 585 nm emission.
Question 2: What is the labeling stoichiometry?
Answer 2: Rhodamine labeling stoichiometry was calculated to be 1-3 dyes per fibronectin protein using the absorbance maximum for rhodamine at 565 nm and the Beer-Lambert law. Dye extinction coefficient when protein bound is 70,000 M-1cm-1.
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