Peroxidase Labeling Kit

• Quick: only 3 hours to get conjugates
  
• Easy: all processes in a single filtration tube
  
• Reliable: high recovery of conjugates
  
• Efficient: applicable for 50-200 ug IgG*
  

   

Dojindo offers two types of Peroxidase labeling kit. One is NH2-reactive peroxidase labeling kit (NH2 type) and other is SH-reactive peroxidase labeling kit (SH type). NH2 type reacts with a primary or secondary amine group of target molecules, and SH type reacts with thiol groups. The reaction mechanism of NH2 type and SH type are illustrated below. The labeling process of NH2 type is very simple, and the sensitivity of the peroxidase conjugate is sufficient for most of the detections or assays. However, the sensitivity of the peroxidase-conjugated IgG prepared by SH type kit is usually higher than the one prepared by NH2 type kit because of the site specific labeling reaction. The SH type labeling requires a reducing reaction to create SH group(s) prior to label with SH-reactive peroxidase. If the sensitivity of the peroxodase conjugate is the most important issue, use the SH type kit.

Peroxidase Labeling Kit - NH2 is for simple and rapid preparation of peroxidase-labeled IgG in enzyme immunoassays (EIA) and immunoblotting/immunostaining. It can also be used for preparation of peroxidase-labeled antigen in competitive EIA. NH2-reactive peroxidase (a component of this kit) has an activated ester group, 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 purified with Filtration tube included in this kit. Filtration tube is also used for sample IgG in removing small molecules such as sodium azide, Tris buffer and amine compounds that interfere with the assay or labeling reaction. This kit contains all of the necessary reagents for peroxidase labeling, including the storage buffer for conjugates.

If the IgG solution contains other proteins with molecular weight larger than 10,000, such as serum albumin or gelatin, purify the IgG solution prior to label peroxidase with this kit. Commercially available antibody may contain BSA or gelatin as a stabilizer. Dojindo offers IgG Purification Kit-A (AP01-10) and IgG Purification Kit-G (AP02-10) for the purification of the IgG solution.

Peroxidase Labeling Process to IgG:

HLPC Data of Peroxidase-conjugated IgG:


Protein: rabbit IgG, 100 µg
 HPLC Conditions:
           column: TSKgel column, G3000 PWXL, 30 cm x 7.8 mm, Supelco
           flow rate: 0.8 ml/min
           mobile phase: 10 mM phosphate buffer, pH 7.0
           oven temperature: 28 ºC
           sample volume: 20 µl


FAQ

1. Can I use this kit for other proteins or peptides?
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 greater than 50,000, follow the labeling protocol for IgG, and use 0.5-1 nmol of sample protein. 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 info@dojindo.com or 1-877-987-2667 before purchasing the kit.

2. Can I use this kit to label an oligonucleotide?
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.

3. 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. Ten µg IgG can still be labeled using this kit, but the background will be higher.

4. How many HRP molecules per IgG are introduced?
An average of 1 to 3 peroxidase molecules per IgG.

5. Does unconjugated NH2-reactive peroxidase still have an activated ester after the labeling reaction to IgG?
No. NHS is completely hydrolyzed during the reaction.

6. 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.

7. Do I have to use Storage Buffer included with the kit?
No, you do not have to use Storage buffer from this kit. You can choose any kind of buffer appropriate for your experiment.

Ordering Information