Fibronectin (Green fluorescent, HiLyte 488)

Fibronectin (Green fluorescent, HiLyte 488)

Product Uses Include

  • Observation of fibronectin matrix assembly and cell adhesion
  • Cell invasion assays (1)
  • FACS analysis

Fibronectin is purified from bovine plasma.  Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% polyacrylamide gel.  HiLyte Fluor™ 488 labeled fibronectin is >80% pure (Figure 1). 

The protein is modified to contain covalently linked HiLyte Fluor™ 488 at random surface lysines (2). An activated ester of the fluorochrome is used to label the protein. Labeling stoichiometry is determined by spectroscopic measurement of protein and dye concentrations. Final labeling stoichiometry is 1-3 dyes per protein molecule (Figure 2). HiLyte Fluor™ 488 labeled fibronectin can be detected using a filter set of 350-450nm excitation and 500-550 nm emission.

Fibronectin runs as individual subunits on SDS-PAGE with an apparent molecular weight of 230 kDa.  FNR02 is supplied as an orange lyophilized powder.  Each vial of FNR02 contains 20 µg protein.

Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are >80% pure. 


Figure 1:  HiLyte Fluor™ 488 labeled Fibronectin Purity Determination

Legend: 20 µg of unlabeled fibronectin (Lane 1) and 20 µg of HiLyte Fluor™ 488 labeled fibronectin (Lane 2) was separated by electrophoresis in a 4-20% SDS-PAGE system.  The unlabeled protein was stained with Coomassie Blue and visualized in white light. The HiLyte Fluor™ 488 labeled protein was visualized under UV light, no free dye was observed in the dye front. Protein quantitation was determined with the Precision Red™ Protein Assay Reagent (Cat. # ADV02).  Mark12 molecular weight markers are from Invitrogen


Figure 2:   Absorption scan of HiLyte Fluor™ 488 labeled fibronectin in solution

Legend:  FNR02 was diluted with Milli-Q water and its absorbance spectrum was scanned between 250 and 650 nm.  HiLyte Fluor™ 488 labeling stoichiometry was calculated to be 1-3 dyes per fibronectin protein using the absorbancy maximum for  at 527 nm and the Beer-Lambert law.  Dye extinction coefficient when protein bound is 70,000M-1cm-1


  1. Artym VV. Et al. 2009. ECM degradation assay for analyzing local cell invasion.  Methods in molecular biology, Extracellular matrix protocols, vol. 522: 211-219.Humana Press. 
  2. Use of this product employs the following patent rights licensed to Cytoskeleton, Inc. from Anaspec, Inc.: (a) the claims of U.S. Patents No. 7,754,893, 7,820,783 and 7,790,394; (b) any claims issuing from U.S. Patent Applications Serial No. 12/804,065, 12/807,268 and 12/925,505; and (c) all patents to be issued pursuant thereto, and all continuations, continuations –in-part, reissues, substitutes, and extensions thereof. The use of this product is limited to the field of use comprising internal use by an end user of this product solely in in vivo and in vitro cell staining or biochemical assay applications, such as IHC, HCS, FACS, in vitro assays of an end user only for scientific R&D purposes. The filed of use of this product explicitly excludes the following actions: (a) generating data from clinical applications in humans and animals; and (b) generating QC or QA data for the validation of health, food or cosmetic products.

For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at

AuthorTitleJournalYearArticle Link
Li, Wenhong et al.Differential cellular responses to adhesive interactions with galectin-8- And fibronectin-coated substratesJournal of Cell Science2021ISSN 1477-9137
Pal, Kaushik et al.Ubiquitous membrane-bound DNase activity in podosomes and invadopodiaJournal of Cell Biology2021ISSN 1540-8140
Han, Zuoning et al.Integrin aVβ1 regulates procollagen i production through a non-canonical transforming growth factor β signaling pathway in human hepatic stellate cellsBiochemical Journal2021ISSN 1470-8728
Rong, Zhouyi et al.Activation of FAK/Rac1/Cdc42-GTPase signaling ameliorates impaired microglial migration response to Aβ42 in triggering receptor expressed on myeloid cells 2 loss-of-function murine modelsFASEB Journal2020ISSN 1530-6860
Huang, Yuxing et al.Arp2/3-branched actin maintains an active pool of GTP-RhoA and controls RhoA abundanceCells2019ISSN 2073-4409
Sala, Laura et al.Abrogation of myofibroblast activities in metastasis and fibrosis by methyltransferase inhibitionInternational Journal of Cancer2019ISSN 1097-0215
Foxall, Elizabeth et al.PAK4 Kinase Activity Plays a Crucial Role in the Podosome Ring of Myeloid CellsCell Reports2019ISSN 2211-1247
Stanton, Alice E. et al.Biochemical Ligand Density Regulates Yes-Associated Protein Translocation in Stem Cells through Cytoskeletal Tension and IntegrinsACS Applied Materials and Interfaces2019ISSN 1944-8252
Rafiq, Nisha Bte Mohd et al.Forces and constraints controlling podosome assembly and disassemblyPhilosophical Transactions of the Royal Society B: Biological Sciences2019ISSN 1471-2970
Sun, Xiaoyu et al.Replication of biocompatible, nanotopographic surfacesScientific Reports2018ISSN 2045-2322
Werley, Christopher A. et al.Geometry-dependent functional changes in iPSC-derived cardiomyocytes probed by functional imaging and RNA sequencingPLoS ONE2017ISSN 1932-6203
Kim, Jiyun et al.Three-dimensional patterning of the ECM microenvironment using magnetic nanoparticle self assemblyCurrent Protocols in Cell Biology2016ISSN 1934-2616
Kim, Jiyun et al.Independent Control of Topography for 3D Patterning of the ECM MicroenvironmentAdvanced Materials2016ISSN 1521-4095
Stanisavljevic, Jelena et al.Snail1-expressing fibroblasts in the tumor microenvironment display mechanical properties that support metastasisCancer Research2015ISSN 1538-7445
Torr, Elizabeth E. et al.Myofibroblasts exhibit enhanced fibronectin assembly that is intrinsic to their contractile phenotypeJournal of Biological Chemistry2015ISSN 1083-351X
Jacob, Abitha et al.Rab40b regulates trafficking of MMP2 and MMP9 during invadopodia formation and invasion of breast cancer cellsJournal of cell science2013ISSN 1477--9137
Lively, Starlee et al.The microglial activation state regulates migration and roles of matrix-dissolving enzymes for invasionJournal of Neuroinflammation2013ISSN 1742-2094


Question 1:  What is the optimal excitation and emission filter settings to visualize the HiLyte Fluor™ 488 fluorescence?

Answer 1: HiLyte Fluor™ 488 labeled-fibronectin can be detected using a filter set of 502 nm excitation and 527 nm emission.


Question 2:  What is the labeling stoichiometry?

Answer 2: HiLyte Fluor™ 488 labeling stoichiometry was calculated to be 1-3 dyes per fibronectin protein using the absorbancy maximum for HiLyte 488 at 527 nm and the Beer-Lambert law.  Dye extinction coefficient when protein bound is 70,000 M-1cm-1.



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