标签：Peroxidase Labeling Kit-NH2

Dojindo,过氧化物酶标记试剂盒-NH2/1/LK11,Peroxidase Labeling Kit-NH2

过氧化物酶标记试剂盒-NH2 主要用于制备过氧化物酶标记的 IgG 用于酶免疫分析 (EIA) 和制备过氧化物酶标记的抗原用于竞争性 EIA。 NH2 反应性过氧化物酶是该试剂盒的一个组成部分，具有琥珀酰亚胺基 (NHS)，可与结构中具有氨基的蛋白质或其他分子发生反应（图 1）。该试剂盒包含标记过程所需的所有试剂，包括储存缓冲液。标记过程很简单：将 IgG 与 NH2 反应性过氧化物酶混合并在 37ºC 下孵育 2 小时。 NH2 反应性过氧化物酶无需任何激活过程即可与靶分子形成共价键。 NHS 与过氧化物酶的距离约为 1.2 nm，是过氧化物酶分子半径的一半。因此，当过氧化物酶标记的IgG用于EIA时，NH2反应性过氧化物酶的标记效率足够高，可以省去标记后的纯化过程。此外，过氧化物酶标记不会影响目标分子的亲和力。如果标记后需要高纯度偶联物，只需使用亲和柱或凝胶渗透柱即可。标记小分子时，可以使用该套件中包含的过滤管去除多余的分子。因为 NH2 反应性过氧化物酶的氨基被封闭，所以不可能进行自结合。

Fig.1 IgG labeling reaction of NH2-reactive peroxidase

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 peroxidase-conjugated IgG is always on the membrane of the filtration tube during the labeling process.♦ If the IgG solution contains other proteins with a molecular weight greater than 10,000, such as BSA or gelatin, purify the IgG solution before labeling peroxidase 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 labeling.

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Sandwich ELISA

Fig. 2 Sandwich ELISA of CAT (chloramphenicol acetyl transferase) assay.

Plate: 2 μg/ml anti-CAT antibody (rabbit anti sera)-coated high binding plateCAT: 0-400 x 10-3units/ml PBSTPeroxidase-conjugated anti-CAT antibody: Prepared by Peroxidase Labeling Kit-NH2.1μg/ml PBST+blocking reagentSubstrate: TMB peroxidase substrate

Western blot

Fig. 3 Western blot using peroxidase-labeled monoclonal antibody to SIV p24 Gag(2F12).

SIV P55 and molecular weight markers were analyzed in blot 1, 2, and 3.Blot 1: conjugate prepared using Peroxidase Labeling Kit-NH2Blot 2: conjugate prepared using Peroxidase Labeling Kit-SHBlot 3: primary antibody and peroxidase-conjugated secondary antibody (commercially available).

The western blotting using peroxidase-labeled primary antibody gives abetter result than using peroxidase-labeled secondary antibody. In most cases, the sensitivity of the conjugate prepared with Peroxidase/ Alkaline phosphatase Labeling Kit-SH is higher than Labeling Kit-NH2 due to the site specific conjugation on the antibody.

Can I use this kit for Fab or Fab Elabeling?

Yes, you can label Fab or Fab Eusing this kit. The recovery of the conjugate should be over 80%.

Can I use this kit for other proteins?

Yes, if the molecular weight is greater than 50,000 or less 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 LK11-10.If the molecular weight is less than 5,000, follow the labeling protocol for small molecules. If the molecular weight is higher than 5,000 but lower than 50,000, 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 LK11-10?

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 peroxidase molecules per IgG are introduced?

The average number of peroxidase molecule per IgG is 1 to 3.

Does unconjugated NH2-reactive peroxidase still have an activated ester after the labeling reaction to IgG?

No. It is completely hydrolyzed during the reaction.

Does NH2-reactive peroxidase form an oligomer during the labeling reaction?

No. Since all amino groups of NH2-reactive peroxidase 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.However, the Storage buffer helps to increase the stability of the peroxidase conjugate.

Does Storage buffer contain animal products or polymers?

No, Storage buffer does not contain any animal products, polymers, or heavy metal ions.