Peroxidase Labeling Kit-NH2 is used mainly for the preparation of peroxidase-labeled IgG for enzyme immunoassay (EIA) and for the preparation of peroxidase-labeled antigen for competitive EIA. NH2-reactive peroxidase, a component of this kit, has succinimidyl groups (NHS) and reacts with proteins or other molecules that have an amino group in their structures (Fig. 1). This kit contains all the reagents necessary for the labeling process, including storage buffer. The labeling process is simple: mix IgG with NH2-reactive peroxidase and incubate at 37ºC for 2 hours. The NH2-reactive peroxidase forms a covalent link with the target molecule without any activation process. The distance of the NHS from peroxidase is about 1.2 nm, half of the radius of the peroxidase molecule. Therefore, when the peroxidase-labeled IgG is used for EIA, the labeling efficiency of the NH2-reactive peroxidase is high enough to eliminate the purification process after labeling. Also, peroxidase labeling will not affect the affinity of the target molecule. 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. Because the amino groups of NH2-reactive peroxidase are blocked, no self-conjugation is possible.
Fig.1 IgG labeling reaction of NH2-reactive peroxidase
♦ 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 peroxidase-conjugated IgG is always on the membrane of the filtration tube during the labeling process.
♦ If the IgG solution contains other proteins with a molecular weight greater than 10,000, such as BSA or gelatin, purify the IgG solution before labeling peroxidase 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 labeling.
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Fig. 2 Sandwich ELISA of CAT (chloramphenicol acetyl transferase) assay.
Plate: 2 μg/ml anti-CAT antibody (rabbit anti sera)-coated high binding plate
CAT: 0-400 x 10-3units/ml PBST
Peroxidase-conjugated anti-CAT antibody: Prepared by Peroxidase Labeling Kit-NH2.1μg/ml PBST+blocking reagent
Substrate: TMB peroxidase substrate
Fig. 3 Western blot using peroxidase-labeled monoclonal antibody to SIV p24 Gag(2F12).
SIV P55 and molecular weight markers were analyzed in blot 1, 2, and 3.
Blot 1: conjugate prepared using Peroxidase Labeling Kit-NH2
Blot 2: conjugate prepared using Peroxidase Labeling Kit-SH
Blot 3: primary antibody and peroxidase-conjugated secondary antibody (commercially available).
The western blotting using peroxidase-labeled primary antibody gives abetter result than using peroxidase-labeled secondary antibody. In most cases, the sensitivity of the conjugate prepared with Peroxidase/ Alkaline phosphatase Labeling Kit-SH is higher than Labeling Kit-NH2 due to the site specific conjugation on the antibody.