Allophycocyanin Labeling Kit-NH2

• 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

Phycobiliproteins are fluorescent proteins derived from cyanobacteria and eukaryotic algae. Their fluorescence is much higher than chemical fluorescent probes such as fluorescein and rhodamine. Because of this high fluorescence, phycobiliprotein labeled antibodies and other molecules can give greater sensitivity in flow cytometry and immunoblotting/ immunostaining. Allophycocyanin (APC) is one of the phycobiliproteins, and it has a blue fluorescence at arount 660 nm.

Dojindo offers two types of APC labeling kit. One is NH2-reactive APC labeling kit (NH2 type) and other is SH-reactive APC 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 APC conjugate is sufficient for most of the detections or assays. However, the sensitivity of the APC-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 APC. If the sensitivity of the APC conjugate is the most important issue, use the SH type kit.

NH2-reactive APC (a component of this kit) has activated ester groups, and can easily make a covalent bond with an amino group of the target molecule without any activation process. Filtration tube in this kit allows a quick buffer exchange and concentration of sample IgG solution. This kit contains all of the necessary reagents for APC labeling, including the storage buffer for conjugate.

SH-reactive APC (a component of this kit) has maleimide groups, and can easily make a covalent bond with a sulfhydryl group of the target molecule without any activation process. Filtration tube in this kit allows a quick buffer exchange and concentration of sample IgG solution. This kit contains all of the necessary reagents for APC labeling, including the reducing agent for the preparation of reduced IgG that has SH groups and the storage buffer for conjugate.

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 APC with this kit. Commercially available antibody may contain BSA or gelatin as a stabilizer. Dojindo offers IgG Prep Kit-A (AP01-10) and IgG Prep Kit-G (AP02-10) for the purification of the IgG solution.


Allophycocyanin Labeling Process to IgG:




Fluorescent Spectrum and Intensity:

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. Follow the protocol for IgG labeling with 0.5-1 nmol of sample protein to be labeled.

2. How many Allophycocyanin molecules per IgG are introduced?
An average number of Allophycocyanin molecules is 1 to 2 per IgG.

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

4. Does NH2-reactive Allophycocyanin form an oligomer during the labeling reaction?
No. Since all amino groups of NH2-reactive Allophycocyanin are blocked, no oligomerization is possible.

5. Do I have to use WS Buffer included with the kit?
Yes. Use WS buffer to prepare a stock solution of the conjugate. However, you can choose any kind of buffer appropriate to dilute the stock solution for your experiment.

6. How long is the conjugate stable?
If you store at 4 C, it is stable for over 2 months. For longer strage, add 100% volume of glycerol, aliquot and store at -20 C. However, please note that the stability depends on the protein itself.

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