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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. 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
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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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