Alkaline Phosphatase Labeling Kit-SH is used mainly for the preparation of alkaline phosphatase-labeled IgG for enzyme immunoassay (EIA) and for the preparation of alkaline phosphatase-labeled antigen for competitive EIA. SH-reactive ALP, a component of this kit, can react with the thiol groups of proteins or other molecules (Fig.1). This kit contains all the reagents necessary for the labeling process, including reducing agent and storage buffer. SH-reactive ALP forms a covalent link with the target molecule. Reducing agent can create free thiol groups in the IgG molecule. The labeling efficiency of the SH-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 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 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.
Fig. 2 Sandwich ELISA of human TNF-α detection
Plate: 2μg/ml anti-human TNF-antibody (rabbit, polyclonal)-coated
high binding plate
recombinant human TNF-a: 0-1000 pg/ml PBST
ALP-conjugated anti-human TNF-antibody: Prepared by Alkaline phosphatase Labeling Kit-SH .1μg/ml PBST+blocking reagent
Substrate: p-Nitrophenylphosphate, ALP substrate