HiLyte Fluor  Labeling Kit

• 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
  

HiLyte Fluor*  Labeling Kit-NH2 is mainly used for the preparation of HiLyte Fluor labeled proteins such as IgG for immunostaining and cellular proteins for tracing. NH2-reactive HiLyte Fluor, a component of this kit, has a succinimidyl group (NHS) that reacts with an amino group of proteins or other macromolecules. This kit contains all of the necessary reagents for labeling. Each tube of NH2-reactive HiLyte Fluor can label up to 200 µg of IgG, conjugating about 4 to 6 HiLyte Fluor molecules per IgG molecule. The labeling process is simple. Add the NH2-reactive HiLyte Fluor  to IgG solution on a filter membrane, and incubate at 37 ºC for 10 min. The excess HiLyte Fluor molecules can be removed by a Filtration tube.

HiLyte Fluor Labeling Kit-SH is mainly used for the preparation of HiLyte Fluor-labeled proteins such as IgG for immunostaining and cellular proteins for tracing. SH-reactive HiLyte Fluor , a component of this kit, has a maleimide group that reacts with sulfhydryl group (SH) of proteins or other macromolecules. This kit contains all of the necessary reagents for labeling. Reducing agent in this kit creates free sulfhydryl groups in the IgG molecule without loss of antibody affinity. Each tube of SH-reactive HiLyte Fluor can label up to 200 µg of IgG, conjugating about 5 to 8 HiLyte Fluor 5molecules per IgG molecule. The labeling process is simple. After the reducing reaction, add SH-reactive HiLyte Fluor  to IgG solution on a filter membrane, and incubate at 37 ºC for 30 min. The excess HiLyte Fluor molecules can be removed by a Filtration tube. The excitation and emission wavelengths of the HiLyte Fluor -labeled IgG are  nm and 570 nm, respectively.

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 HiLyte Fluor 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.
*HiLyte Fluor is a trademark of AnaSpec, Inc.

HiLyte Fluor Labeling Reaction

HiLyte Fluor  Labeling Process to IgG:



Determination of HiLyte Fluor/IgG Ratio:

For example of HiLyte Fluor 555, Measure the absorbance of the HiLyte Fluor 555-labeled IgG solution at 280 nm and 555 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 HiLyte Fluor 555. 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 HiLyte Fluor -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 HiLyte Fluor -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. For less than 20 µg, follow the manual and add 4 micro;l NH2-reactive HiLyte Fluor  instead of 8 µl at step 4.

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

Ordering Infomation