Product Uses Include
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
|Xia, Qiaoxi et al.||A protein complex of LCN2, LOXL2 and MMP9 facilitates tumour metastasis in oesophageal cancer||Molecular Oncology||2023||ISSN 1878--0261|
|Baschieri, Francesco et al.||Fibroblasts generate topographical cues that steer cancer cell migration||Science Advances||2023||ISSN 2375-2548|
|Li, Wenhong et al.||Differential cellular responses to adhesive interactions with galectin-8- And fibronectin-coated substrates||Journal of Cell Science||2021||ISSN 1477-9137|
|Pal, Kaushik et al.||Ubiquitous membrane-bound DNase activity in podosomes and invadopodia||Journal of Cell Biology||2021||ISSN 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 cells||Biochemical Journal||2021||ISSN 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 models||FASEB Journal||2020||ISSN 1530-6860|
|Sala, Laura et al.||Abrogation of myofibroblast activities in metastasis and fibrosis by methyltransferase inhibition||International Journal of Cancer||2019||ISSN 1097-0215|
|Foxall, Elizabeth et al.||PAK4 Kinase Activity Plays a Crucial Role in the Podosome Ring of Myeloid Cells||Cell Reports||2019||ISSN 2211-1247|
|Stanton, Alice E. et al.||Biochemical Ligand Density Regulates Yes-Associated Protein Translocation in Stem Cells through Cytoskeletal Tension and Integrins||ACS Applied Materials and Interfaces||2019||ISSN 1944-8252|
|Huang, Yuxing et al.||Arp2/3-branched actin maintains an active pool of GTP-RhoA and controls RhoA abundance||Cells||2019||ISSN 2073-4409|
|Rafiq, Nisha Bte Mohd et al.||Forces and constraints controlling podosome assembly and disassembly||Philosophical Transactions of the Royal Society B: Biological Sciences||2019||ISSN 1471-2970|
|Sun, Xiaoyu et al.||Replication of biocompatible, nanotopographic surfaces||Scientific Reports||2018||ISSN 2045-2322|
|Werley, Christopher A. et al.||Geometry-dependent functional changes in iPSC-derived cardiomyocytes probed by functional imaging and RNA sequencing||PLoS ONE||2017||ISSN 1932-6203|
|Kim, Jiyun et al.||Three-dimensional patterning of the ECM microenvironment using magnetic nanoparticle self assembly||Current Protocols in Cell Biology||2016||ISSN 1934-2616|
|Kim, Jiyun et al.||Independent Control of Topography for 3D Patterning of the ECM Microenvironment||Advanced Materials||2016||ISSN 1521-4095|
|Torr, Elizabeth E. et al.||Myofibroblasts exhibit enhanced fibronectin assembly that is intrinsic to their contractile phenotype||Journal of Biological Chemistry||2015||ISSN 1083-351X|
|Stanisavljevic, Jelena et al.||Snail1-expressing fibroblasts in the tumor microenvironment display mechanical properties that support metastasis||Cancer Research||2015||ISSN 1538-7445|
|Jacob, Abitha et al.||Rab40b regulates trafficking of MMP2 and MMP9 during invadopodia formation and invasion of breast cancer cells||Journal of cell science||2013||ISSN 1477--9137|
|Lively, Starlee et al.||The microglial activation state regulates migration and roles of matrix-dissolving enzymes for invasion||Journal of Neuroinflammation||2013||ISSN 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.
If you have any questions concerning this product, please contact our Technical Service department at firstname.lastname@example.org