Alkaline Phosphatase Labeling Kit-NH2 is mainly used for the preparation of alkaline phosphatase-labeled IgG for enzyme immunoassay (EIA) and for the preparation of alkaline phosphataselabeled antigen for competitive EIA. NH2-reactive ALP, a component of this kit, can react with amino groups of proteins or other molecules (Fig. 1-15). This kit contains all of the necessary reagents for the labeling process including Storage buffer. NH2-reactive ALP forms a covalent link with the target molecule without any activation process. The labeling efficiency of the NH2-reactive ALP is high enough to eliminate any purification process after labeling when the alkaline phosphatase-labeled IgG is used for EIA. If a high purity conjugate is required after labeling, simply use an affinity column or a gel permeation column. When labeling small molecules, excess molecules can be removed by using the Filtration tubes included in this kit.
Fig. 1 IgG labeling reaction of NH2-reactive alkaline phosphatase
♦ The molecular weight of the protein to be labeled with this kit should be greater than 50,000.
♦ The molecular weight of the small amine compound to be labeled with this kit should be smaller than 5,000.
♦ IgG or alkaline phosphatase-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 larger than 10,000, such as BSA or gelatin, purify the IgG solution prior to labeling alkaline phosphatase with this kit. IgG solution can be purified by IgG Purification Kits (not included in this kit).
♦ If the IgG solution contains small insoluble materials, centrifuge the solution and use the supernatant for the labeling.
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Fig. 2 Western blotting of Alkaline phosphatase-conjugated antibody prepared by Alkaline phosphatase Labeling Kit-NH2.
1: antigen 50 ng
2: antigen 10 ng
3: antigen 2 ng
A target protein (antigen) was detected with ALP-labeled antibody prepared by Alkaline Phosphatase Labeling Kit-NH2 after it was run with SDS-PAGE and transferred to a nitrocellulose membrane. A target protein was detected with a chemiluminescence substrate for alkaline phosphatase after the treatment with 25,000 times dilution of ALP-labeled primary antibody.