Fluorescein Labeling Kit-NH2 is mainly used for the preparation of fluorescein-labeled proteins such as IgG for immunostaining and cellular proteins for tracing. Amine-reactive fluorescein, a component of this kit, has succinimidyl groups (NHS) that react with the amino groups on proteins or other molecules (Fig. 1). This kit contains all the reagents necessary for labeling, including storage buffer. Each vial of fluorescein can label up to 200 μg of IgG, conjugating about 4 to 6 fluorescein molecules per IgG molecule. Because this kit also includes a buffer exchange system, a sample containing amine base buffer can be labeled. Although membrane filtration sometimes causes IgG aggregation, the buffer system in this kit prevents aggregation during the concentration of IgG or fluorescein-labeled IgG solution. A fluorescein-labeled IgG solution prepared using this kit is stable for more than 2 months at 4ºC. The excitation and emission wavelengths of the fluoresceinlabeled IgG are 495 nm and 520 nm, respectively (Fig. 2).
Fig. 1 IgG labeling reaction of NH2-reactive fluorescein
Fig. 2 Fluorescence spectrum of fluorescein-conjugated IgG
♦ The molecular weight of the protein to be labeled with this kit should be greater than 50,000.
♦ IgG or fluorescein-conjugated IgG is always on the membrane of the filtration tube during the labeling process.
♦ If the IgG solution contains other proteins with molecular weight greater than 10,000, such as BSA or gelatin, purify the IgG solution before labeling fluorescein with this kit. IgG solution can be purified by IgG Purification Kits (not included in this kit).
♦ If the IgG solution contains small insoluble material, centrifuge the solution and use the supernatant for the labeling.
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