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.
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
Q. Can I use this kit for F(ab E2?
A. Yes, please follow the labeling protocol for IgG. The recovery of the conjugate should be over 80%.
Q. Can I use this kit for other proteins or peptides?
A. Yes, if the molecular weight of the reduced form is greater than 50,000 or less than 5,000, and it has a reactive SH group or a disulfide group that can be reduced without losing activity. If the molecular weight is greater than 50,000, follow the labeling protocol for IgG and use 0.5-1 nmol of sample protein for LK13-10. If the molecular weight is less than 5,000, follow the labeling protocol for small molecules. If the molecular weight is between 5,000 and 50,000, contact our customer service at firstname.lastname@example.org or 1-877-987-2667 for more information.
Q. Can I use this kit to label oligopeptides or oligonucleotides?
A. Yes, if the molecular weights of the oligonucleotide or the oligopeptide are less than 5,000 and they have at least one SH group. Follow the labeling protocol for small molecule.
Q. What is the minimum amount of IgG that can be labeled with LK13-10?
A. The minimum amount is 50 μg. There is no significant difference in sensitivity and background between 50 μg and 200 μg of IgG. However, even 10 μg IgG can be labeled, using 1/5 volume of SH-reactive ALP solution at step 8.
Q. How many alkaline phosphatase molecules per reduced IgG are introduced?
A. The average number of alkaline phosphatase molecule per reduced IgG is 1 to 2.
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 reactive SH groups 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 Solution B and add the mixture to a vial of the SH-reactive peroxidase.
Q. Do I have to use Storage buffer included with the kit?
A. No, you don’t have to use Storage buffer from the kit. You can choose any kind of buffer appropriate for your experiment.
Q. My sample contains small insoluble material. What should I do?
A. Spin the sample and use the supernatant for labeling.
Q. Does unconjugated SH-reactive ALP still have a reactive maleimide after the labeling reaction to IgG?
A. No. Nearly 100% of SH-reactive ALP is used for the IgG labeling or the small molecule labeling.
Q. Does Storage buffer contain animal products or polymers?
A. No, Storage buffer does not contain any animal products , polymers, or heavy metal ions.