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).
IgG labeling reaction of NH2
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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Q. Can I use this kit for other proteins?
A. Yes, if the molecular weight is greater than 50,000.
Q. Do I have to use a Filtration tube prior to labeling the protein?
A. If the protein solution does not contain small molecules with an amino group and the concentration of the protein is 10 mg per ml, or about 70 μM, there is no need to use the Filtration tube. Just mix 10 μl of the sample solution with 90 μl of Reaction buffer and add 8 μl NH2-reactive fluorescein (prepared at step 3) to the mixture, and follow the protocol starting at step 4.
Q. How long is the fluorescein-labeled protein stable?
A. If you store at 4ºC, it is stable for over 2 months. For longer storage, add 100% volume of glycerol, aliquot, and store at -20ºC. However, please note that stability depends on the protein itself.
Q. What is the minimum amount of IgG that can be labeled by this kit?
A. The minumum amount of IgG is 10 μg; simply follow the protocol. The labeling ratio remains the same for 10 μg to 100 μg of IgG.
Q. Can I use this kit to label oligonucleotides or peptides?
A. No. Oligonucleotides and peptides may be too small to retain on the membrane filter of the Filtration tube.