Biotin 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
  

   

Biotin Labeling Kit-NH2 is primarily used for the preparation of biotin-labeled IgG for enzyme immunoassay (EIA). NH2-reactive biotin, a component of this kit, has a succinimidyl group (NHS) that reacts with an amino group of IgG and other macromolecules. This kit contains all of the necessary reagents for labeling including the storage buffer. The labeling process is simple. Just add the NH2-reactive biotin to IgG solution on a filter membrane, and incubate at 37 ºC for 10 min. On the average, 5 to 8 biotin molecules conjugate to each IgG molecule. Excess biotin molecules can be removed using a filtration tube included in this kit. Biotin Labeling Kit-SH (LK10-10) is also available.

Biotin Labeling Kit-SH is primarily used for the preparation of biotin-labeled IgG for enzyme immunoassay (EIA). SH-reactive biotin, a component of this kit, has a maleimide group that reacts with sulfhydryl groups of reduced IgG or other macromolecules. This kit contains all of the necessary reagents for the labeling including the storage buffer. Reducing agent in this kit creates free sulfhydryl groups in the IgG molecule without loss of affinity. The labeling process is simple. After the reducing reaction, add SH-reactive biotin to IgG solution on the filter membrane, and incubate at 37ºC for 30 min. On the average, 5 to 8 biotin molecules conjugate to each IgG molecule. Excess biotin molecules can be removed using a filtration tube included in this kit. Biotin Labeling Kit-NH2 (LK03-10) is 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 biotin 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.

Biotin Labeling Reaction

Biotin Labeling Process to IgG:



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 groups and the concentration of the protein is 10 mg/ml or about 70 µM, there is no need to use the filtration tube. Mix 10 µl of the sample solution with 90 µl of Reaction buffer, and add 8 µl NH2-reactive biotin solution (prepared in Step 3) to the mixture, and follow the protocol starting at Step 4.

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

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

5. How can I determine the number of biotin per protein?
The average number of biotin per IgG should be in the range of 5 and 8. If you need to determine precise number of biotin molecule per protein, use HABA assay. Following is a HABA assay protocol

Reagent solution

prepare various concentration biotin solutions (12.5 µM, 6.25 µM, 3.13 µM, 1.56 µM) (200 µl each)

1. Mix HABA solution and avidin solution in a plastic tube.br> 2. Add 100 µl of the HABA-avidin solution to 15 wells    for multiple assays (n=3).
3. Add 50 µl biotin solution (12.5 µM, 6.25 µM, 3.13 µM, 1.56 µM) to 3 wells each and 50 µl of diluted sample    solution to the rest of the 3 wells.
4. Read the O.D. at 405 nm with a reference at 492 nm, and prepare a calibration curve using the O.D. of various    concentration of biotin solutions. Read the O.D. at 280 nm to determine the protein concentration (e.g. molar    absorptivity of IgG at 280 nm: 216,000).
5. Determine the concentration of biotin in the sample solution, and calculate the number of biotin molecule per    protein.

Ordering Information