Peroxidase Labeling Kit-SH 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. SH-reactive peroxidase, a component of this kit, can react with the thiol groups of proteins or other molecules (Fig. 1). This kit contains all the necessary reagents for the labeling process, including reducing agent and storage buffer. SH-reactive peroxidase forms a covalent link with the target molecule. Reducing agent can create free thiol groups in the IgG molecule. When peroxidase-labeled IgG is used for EIA, the labeling efficiency of the SH-reactive peroxidase is high enough to eliminate any postlabeling purification process.. If a high-purity conjugate is required after labeling, simply use an affinity column or a gelpermeation 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 SH-reactive peroxidase
♦ The molecular weight of the reduced protein to be labeled with this kit should be greater than 50,000.
♦ The molecular weight of the small thiol 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 molecular weight larger than 10,000, such as BSA or gelatin, purify the IgG solution prior to 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 the labeling.
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Fig. 2 Sandwich ELISA of human TNF-α detection
Plate: 2 μg/ml anti-human TNF-aantibody (rabbit, polyclonal)-coated high binding plate
recombinant human TNF-a: 0-1000 pg/ml PBST
Peroxidase-conjugated anti-human TNF-aantibody: Prepared by Peroxidase Labeling Kit-SH.1μg/ml
Substrate: TMB peroxidase substrate