Alkaline Phosphatase Labeling Kit - NH₂LK12 Product Manual

Alkaline Phosphatase Labeling Kit - NH₂ is for simple and rapid preparation of alkaline phosphatase-labeled IgG in enzyme immunoassay (EIA) and immunoblotting / immunostaining. It can also be used for preparation of alkaline phosphatase-labeled antigen in competitive EIA. NH₂-Reactive Alkaline Phosphatase (a component of this kit) has succinimidyl ester groups, and can easily make a covalent bond with an amino group of the target molecule without any activation process. If the target is a small molecule, the conjugate can be purifi ed with Filtration Tube included in this kit. Filtration Tube is also used for sample IgG in removing small molecules such as Tris buffer and amine compounds that interfere with the assay or labeling reaction. This kit contains all of the necessary reagents for alkaline phosphatase labeling, including the storage buffer for conjugates.

NH2-Reactive Alkaline Phosphatase	3 tubes
Reaction Buffer	200 μl x 1
Washing Buffer	4 ml x 1
Storage Buffer	4 ml x 1
Filtration Tube	3 tubes

Three samples labeling
- Sample requirement: Protein (Molecular weight > 50,000; amount: 50-200 μg)
                                  Small molecule (Molecular weight < 5,000)

Store at 0-5 oC. This kit is stable for 1 year at 0-5^oC before opening.

Caution After a NH2-Reactive Alkaline Phosphatase is taken out from the seal bag, keep the unused NH2-Reactive Alkaline Phosphatase(s) in the bag, seal tightly and store at -20oC. Store the other components at 0-5oC.

• 10 μl, 200 μl adjustable pipettes
• Incubator (37°C)
• Microcentrifuge
• Microtubes

• If the target protein solution contains other proteins with molecular weight larger than 10,000, such as serum albumin or gelatin, purify the protein solution, and use the purifi ed target proteins for alkaline phosphatase labeling, because it might interfere the labeling reaction.
• If the protein solution contains small insoluble materials, centrifuge the solution, and use the supernatant for the labeling.
• The droplets which induced from the dry inhibitor of membrane, are occasionally found inside Filtration Tube while storing the kit at 0-5°C or after returning to room temperature. This phenomenon does not affect the performance.
• This kit includes microtubes containing solutions. Since there is a possibility that the droplets might attach to the inside walls or caps, please shake them down prior to open.

- Labeling for IgG ^a) -

a) The volume of sample solution should be less than 100 μl. If the IgG concentration is lower than 0.5 mg/ml, repeat Steps 1 and 2 until the total IgG accumulation becomes 50-200 μg.

b) If the solution still remains on the membrane after the centrifugation, spin for another 5 min.

c) NH₂-Reactive Alkaline Phosphatase is unstable in Reaction Buffer. Proceed to Step 6 immediately after the preparation of the NH₂-Reactive Alkaline Phosphatase solution.

d) One to three molecules of alkaline phosphatase should be introduced onto one IgG molecule. Unconjugated alkaline phosphatase should not interfere with normal immunoassays. If purifi cation is necessary, use a gel permeation column or an affi nity column for IgG.

e) We recommend using Storage Buffer to recover the conjugate. However, you can use an appropriate buffer for the downstream experiments.

- Labeling for Small Molecule with Amino Group -

a) If the amine compound does not dissolve in aqueous solution, dissolve it with DMSO to prepare 10 mmol/l solution, and mix 5 μl of this solution with 45 μl Reaction Buffer.

b) If the solution still remains on the membrane after the centrifugation, spin for another 5 min.

c) One to two target molecules should be conjugated with one alkaline phosphatase molecule.

d) We recommend using Storage Buffer to recover the conjugate. However, you can use an appropriate buffer for the downstream experiments.

Can I use this kit to label antibody which is commercially available?

Yes. However, if antibody solution contains other proteins such as serum albumin or gelatin, labeling reaction might be interfered by that proteins. Purifi cation of the antibody solution with affi nity chromatography is necessary prior to using this kit. Contact us for the purifi cation procedure, if you need.

How long is the alkaline phosphatase labeled protein stable?

The stability depends on the protein itself. In the case of labeling for goat IgG, the labeled IgG is stable at 4^oC for 2 months. However, for longer storage, please store at -20^oC.

Can I use this kit for other proteins?

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 50-200 μg of sample protein. If it 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.

Can I use this kit to label oligonucleotides or peptides?

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 this kit?

We recommend using 50 μg as a minimum amount. Though 10 μg IgG can still be labeled using this kit, the background will be increased.

Does NH₂-Reactive Alkaline Phosphatase form an oligomer during the labeling reaction?

No. Since all amino groups of NH₂-Reactive Alkaline Phosphatase are blocked, no oligomerization is possible.