Contents of the Kit
||:100 μg x 3 tubes
||:4 ml x 1 bottle
||:200 μl x 1 tube
||:4 ml x 1 bottle
||:1 mg x 1 tube
||:10 ml x 1 bottle
||:1.2 ml x 1 tube
||:10 ml x 1 bottle
||15 ml tube
* ALP: alkaline phosphatase
Storage Condition : 0-5oC
Shipping Condition: with blue ice
Required Equipment and Materials
LK12-10: microcentrifuge, 10 μl and 50-200 μl adjustable pipettes, 37ºC incubator, 0.5 ml microtubes
LK59-10: centrifuge, rotor for 15 ml centrifuge tube, 50-200 μl and 1 ml adjustable pipettes, 37ºC incubator, microtubes
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.
1) B. Pandey, A.V. Demchenko, K.J. Stine, "Nanoporous gold as a solid support for protein immobilization and development of an electrochemical immunoassay for prostate specific antigen and carcinoembryonic antigen", Microchim Acta., 2012, 179, (1-2), 71.
2) M. Watanabe, I. Takemasa, N. Kaneko, Y. Yokoyama, E. Matsuo, S. Iwasa, M. Mori, N. Matsuura, M. Monden, and O. Nishimura, "Clinical significance of circulating galectins as colorectal cancer markers", Oncol. Rep.., 2011, 25, (5), 1217.
3) Y. Matsumae, Y. Takahashi, H. Shiku and T. Matsue, "Quantitative Real‐Time Monitoring of Antibody‐Induced Internalization of Epidermal Growth Factor Receptor on Single Living Mammalian Cells Using Scanning Electrochemical Microscopy", ChemElectroChem., 2018, 5, (20), 3096.
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.
Can I use this kit for Fab or Fab Elabeling?
Yes, you can label Fab and Fab Eusing this kit. The recovery of the conjugate should be over 80%.
Can I use this kit for other proteins or peptides?
Yes, if the molecular weight is higher than 50,000 or lower than 5,000, and it has a reactive primary or secondary amino group. If the molecular weight is higher than 50,000, follow the labeling protocol for IgG, and use 0.5-1 nmol of sample protein for LK12-10. If the molecular weight is lower than 5,000, follow the labeling protocol for small molecules. If the molecular weight is lower than 50,000 but higher than 5,000, contact our customer service at email@example.com or 1-877-987-2667 for more information.
Can I use this kit to label an oligonucleotide or oligopeptide?
Yes, if the molecular weight is less than 5,000, and it has a reactive primary or secondary amino group. Follow the labeling protocol for small molecules.
What is the minimum amount of IgG that can be labeled with LK12-10?
The minimum amount is 50 μg. There is no significant difference in sensitivity and background between 50 μg and 200 μg of IgG. Though 10 μg IgG can still be labeled using this kit, the background will be higher.
How many alkaline phosphatase molecules are introduced per IgG?
The average number of alkakine phosphatase molecule per IgG is 1 to 3.
Does unconjugated NH2-reactive alkaline phosphatase still have an activated ester after the labeling reaction with IgG?
No. NHS is completely hydrolyzed during the reaction.
Does NH2-reactive alkaline phosphatase form an oligomer during the labeling reaction?
No. Since all reactive amino groups of NH2-reactive alkaline phosphatase are blocked, no oligomerization
Do I have to use Storage buffer included with the kit?
No, you do not have to use Storage buffer from the kit. You can choose any kind of buffer appropriate for your experiment.
Does Storage buffer contain animal products or polymers?
No, Storage buffer does not contain any animal products , polymers, or heavy metal ions.