Contents of the Kit
||:4 ml x 1 bottle
||:1 ml x 1 tube
Storage Condition : 0-5ºC
Shipping Condition : ambient temperature
Required Equipment and Materials
microcentrifuge, 10 μl and 100-200 μl adjustable pipettes, 37ºC incubator, 0.5 ml microtubes, DMSO
Biotin Labeling Kit-SH is mainly used for the preparation of biotin-labeled IgG for enzyme immunoassay (EIA). SH reactive biotin, a component of this kit, reacts with sulfhydryl groups of proteins or other molecules (Fig. 1). The kit contains all the reagents necessary for the labeling. The reducing agent included in this kit creates sulfhydryl groups in the IgG molecule. Because the reducing agent is a water-soluble phosphine compound, it can be removed by a filtration tube with a simple process. An average of 5 to 8 biotin molecules conjugate to each IgG molecule. The number of biotin molecules per protein can be determined by HABA assay. Excess biotin molecules can be removed by a filtration tube.
Fig. 1 IgG labeling reaction with SH-reactive biotin
♦ The molecular weight of the reduced protein to be labeled with this kit should be greater than 50,000.
♦ IgG or biotin-conjugated IgG is always on the membrane of the filtration tube during the labeling process.
♦ If the IgG solution contains other proteins with molecular weights larger than 10,000, such as BSA or gelatin, purify the IgG solution before labeling biotin with this kit. IgG solution can be purified by IgG Purification Kits (not included in this kit).
♦ If the IgG solution contains small insoluble materials, centrifuge the solution and use the supernatant for the labeling.
Can I use this kit for other proteins?
Yes, if the molecular weight is greater than 50,000.
Do I have to use a Filtration tube prior to labeling the protein?
There is no need to use the Filtration tube if the following conditions are met.
a) The protein has disulfide groups and its solution does not contain small molecules with SH groups or disulfide groups. The concentration of the protein is 10 mg per ml or about 70 μM:Mix 10 μl protein solution and 100 μl Reducing agent solution, and follow the protocol starting at step 4.
b) The protein has SH groups and its solution does not contain small molecules with SH groups. The concentration of the protein is 10 mg per ml or about 70 μM: Mix 10 μl protein solution and 90 μl Reaction buffer, and add 8 μl SH-reactive biotin solution. Then follow the protocol starting at step 7.
Do I have to use WS buffer to store the biotin-labeled protein?
You don’t have to use WS buffer. You can choose any kind of buffer according to your experiment.
My sample contains small, insoluble material. What should I do?
Spin the sample and use the supernatant for the labeling.
How long is the biotin-labeled protein stable?
If you store the biotin-labeled protein at 4ºC, it is stable for 2 months. For longer storage, add 100% volume of glycerol, aliquot, and store at -20ºC (if the protein can be frozen). However, please note that stability depends on the protein itself.
What is the minimum amount of IgG that can be labeled by this kit?
The minimum amount is 10 μg. There is no significant difference in sensitivity and background between 10 and 100 μg of IgG.