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Fibronectin is purified from bovine plasma. Protein purity is determined by scanning densitometry of coomassie blue stained protein on a 4-20% polyacrylamide gel. Biotinylated fibronectin is >80% pure (Figure 1).
The protein is modified to contain covalently linked biotins at random surface lysines. A long-chain activated ester of biotin [biotin-XX, succinimidyl ester] is used to label the protein. Labeling efficiency is determined by the ability to detect 10 ng biotinylated fibronectin using alkaline phosphatase conjugated streptavidin (Figure 2).
Fibronectin runs as individual subunits on SDS-PAGE with an apparent molecular weight of 230 kDa. FNR03 is supplied as a white lyophilized powder. Each vial contains 20 µg protein.
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are >80% pure.
Figure 1: Fibronectin Purity Determination
Legend: 50 µg of biotinylated fibronectin was separated by electrophoresis in a 4-20% SDS-PAGE system. The protein was stained with coomassie blue. Protein quantitation was determined with the Precision Red™ Protein Assay Reagent (Cat. # ADV02). Mark12 molecular weight markers are from Invitrogen.
Figure 2: Detection of Biotinylated Fibronectin
Legend: Serial dilutions of biotinylated fibronectin were separated by electrophoresis on a 4-20% polyacrylamide gel, blotted to PVDF, probed with a 1:1000 dilution of streptavidin alkaline phosphatase (Sigma) and detected with 1-Step NBT/BCIP reagent™ (Pierce). Lane 1, 100 ng, Lane 2, 50 ng, Lane 3, 40 ng, Lane 4, 20 ng, and Lane 5, 10 ng of biotinylated fibronectin. Lane M, SeeBlue™ molecular weight markers (Invitrogen). Arrow indicates biotinylated fibronectin.
Andrew T. Phillips, Edward F. Boumil, Nileyma Castro, Arunkumar Venkatesan, Eugenio Gallo, Jarrett J. Adams, Sachdev S. Sidhu, Audrey M. Bernstein; USP10 Promotes Fibronectin Recycling, Secretion, and Organization. Invest. Ophthalmol. Vis. Sci. 2021;62(13):15. doi: https://doi.org/10.1167/iovs.62.13.15. (2021)
Varadaraj, A., Magdaleno, C. & Mythreye, K. Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells. BIO-PROTOCOL 8, (2018).
Varadaraj, A. et al. TGF-β triggers rapid fibrillogenesis via a novel TβRII-dependent fibronectin-trafficking mechanism. Mol. Biol. Cell 28, 1195–1207 (2017).
Wang, Q., Da’dara, A. A. & Skelly, P. J. The human blood parasite Schistosoma mansoni expresses extracellular tegumental calpains that cleave the blood clotting protein fibronectin. Sci. Rep. 7, 1–13 (2017).
N.M. Rodriguez et al., 2014. Micropatterned multicolor dynamically adhesive substrates to control cell adhesion and multicellular organization. Langmuir. DOI: 10.1021/la404037s.
Question 1: What is the size of the spacer between the fibronectin and the biotin label?
Answer 1: The biotin label has been modified to contain a 14 atom spacer between the biotin and the fibronectin to improve the accessibility of biotin to avidin/streptavidin.
Question 2: What is the labeling efficiency of the biotinylated fibronectin?
Answer 2: Labeling efficiency is determined by the ability to detect 10 ng of biotinylated fibronectin using alkaline phosphatase-labeled streptavidin.
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