CuV and TuV VLPs were fluorescently labeled using the DyLight 488 antibody labeling kit (Thermo Fisher) following a modified version of the manufacturer’s protocol. A total of 40 μL of borate buffer (0.67 M, pH 8.5) was added to 500 μL of the VLPs at a concentration >0.5 mg/mL, mixed and transferred to the DyLight reagent vial. The mixture was incubated for 1 h at RT protected from light. Unbound fluorescent molecules were removed from the sample by dialysis using a membrane with a 30 kDa cutoff into 4 L of 1× PBS. The dialysis was performed at 4 °C with slow stirring utilizing a magnetic stirrer. The dialysis buffer was changed two additional times after 3 h of dialysis. The success of the labeling procedure was confirmed by SDS-PAGE showing fluorescent VP2 bands when viewed under UV light.
Dylight 488 antibody labeling kit
Manufactured by Thermo Fisher Scientific
The DyLight 488 antibody labeling kit is a tool used for covalently labeling antibodies with the DyLight 488 fluorescent dye. The kit provides the necessary reagents and instructions to perform the labeling process.
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3 protocols using dylight 488 antibody labeling kit
1
Fluorescent Labeling of VLP Samples
2
EGFR-Targeted Binding Assay for Pancreatic Cancer
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Immunofluorescence assay was performed to determine the binding activity of ABD-LDP-Ec to pancreatic cancer cells overexpressing EGFR. The recombinant proteins were pretreated with DyLight 488 Antibody Labeling kit (Thermo Fisher Scientific, Inc.). The proteins diluted in 0.05 M borate buffer were incubated with the DyLight reagent at room temperature for 1 h protected from light. The labeling reaction mixtures were added into the spin columns preloaded with purification resin and mixed with the resin by briefly vortexing, and the columns were centrifuged to collect the labeled proteins. AsPC-1, MIA PaCa-2 and BxPC-3 cells were seeded on coverslips, incubated at 37°C overnight and fixed with 100% methanol at −20°C for 10 min. The cells were incubated with 50 µM (~1 mg/ml) DyLight 488-labeled ABD-LDP-Ec at room temperature for 1 h. Fluorescence was observed under a fluorescence microscope (Nikon Corporation) at ×400 magnification and images were captured in four random fields of view.
3
Fluorescent Labeling of Staphylococcus aureus
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Staphylococcus aureus (strain Newbold) was grown overnight in brain heart infusion (Thermo Scientific, Pierce Biotechnology Inc.) broth at 37°C on a shaker set at 100 rpm. Before labeling, bacteria were washed twice with PBS. For labeling a DyLight 488 Antibody Labeling Kit (Thermo Scientific, Pierce Biotechnology Inc.) was used according to the manufacturer's instruction. After incubation, the bacterial suspension was washed 4 times in PBS to remove unbound label. Bacterial concentration was determined by measuring the optical density at 600 nm and aliquoted in 1.5-mL microtiter tubes. To determine the final concentration of viable bacteria a serial 10-fold dilution was prepared and 50 µL was cultured on sheep blood agar overnight. Aliquots were stored at -20°C.
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