LK12-10 LK59-10
Contents of the Kit
LK12-10
| NH2-reactive ALP | :100 μg x 3 tubes | Washing buffer | :4 ml x 1 bottle |
| Reaction buffer | :200 μl x 1 tube | Storage buffer | :4 ml x 1 bottle |
| Filtration tube | :3 tubes | | |
LK59-10
| NH2-reactive ALP | :1 mg x 1 tube | Washing buffer | :10 ml x 1 bottle |
| Reaction buffer | :1.2 ml x 1 tube | Storage buffer | :10 ml x 1 bottle |
| Filtration tube | :1 tube | 15 ml tube | :1 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
Product Description
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
Precaution
♦ 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.
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 info@dojindo.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
is possible.
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.