Fluorescein Labeling Kit-NH2

• Quick: only 1 hour to get conjugates
  
• Easy: all processes in a single filtration tube
  
• Reliable: high recovery of conjugates
  
• Efficient: applicable for 50-200 ug IgG
  

Fluorescein Labeling Kit-NH2 is mainly used for the preparation of fluorescein-labeled proteins such as IgG for immunostaining and cellular proteins for tracing. NH2-reactive fluorescein, a component of this kit, has a succinimidyl group (NHS) that reacts with an amino group of proteins or other molecules. This kit contains all of the necessary reagents for labeling, including storage buffer. Each tube of NH2-reactive fluorescein can label up to 200 µg of IgG, conjugating about 4 to 6 fluorescein molecules per IgG molecule. The labeling process is simple. Add the NH2-reactive fluorescein to IgG solution on a filter membrane, and incubate at 37 ºC for 10 min. The excess fluorescein molecules can be removed by a Filtration tube. The excitation and emission wavelengths of the fluorescein-labeled IgG are 495 nm and 520 nm, respectively. Red color fluorescent dye labeling kit (Oyster-556 Labeling Kit-NH2: LK04-10) and blue color fluorescent dye labeling kit (Oyster-656 Labeling Kit-NH2: LK07-10) are also available.

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 fluorescein 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.

Fluorescein Labeling Reaction

Fluorescein Labeling Process to IgG:



Determination of Fluorescein/IgG Ratio:

Measure the absorbance of the fluorescein-labeled IgG solution at 280 nm and 500 nm. Calculate the ration using the following equation:






FAQ

1. Can I use this kit for other proteins?
Yes, if the molecular weight is greater than 50,000.

2. Do I have to use a Filtration tube prior to labeling the protein?
If the protein solution does not contain small molecules with amino group and the concentration of the protein is 10 mg/ml, or about 70 µM, there is no need to use the filtration tube. Just mix 10 µl of the sample solution with 90 µl of Reaction buffer and add the mixture to a vial of NH2-reactive fluorescein. After the reaction, transfer all of the reaction mixture to a Filtration tube and follow the protocol starting at Step 7.

3. Do I have to use WS buffer for storing the fluorescein labeled protein?
You do not have to use WS buffer. You can choose any kind of buffers according to your experiment.

4. My sample has small insoluble material. What should I do?
Spin the sample and then use the supernatant for the labeling.

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

6. What is the minimum amount of IgG that can be labeled by this kit?
The minumum amount of IgG is 10 µg; simply follow the same protocol. The labeling ratio remains the same for 10 µg to 100 µg of IgG.

7. Can I use this kit to label oligonucleotides or oligopeptides? No. Oligonucleotides and opeptides may be too small to retain on the membrane filter of the filtration tube.

Ordering Information
NH2-reactive fluorescein : 100 µg x 3 tubes
WS buffer : 4 ml x 1 bottle
Reaction buffer : 0.5 ml x 1 tube
Filtration tube : 3 tubes

Fluorescein Labeling Kit-NH2 Technical Manual