Biotin Labeling Kit-NH2 is mainly used for the preparation of biotin-labeled IgG for enzyme immunoassay (EIA). NH2-reactive biotin, a component of this kit, has succinimidyl groups (NHS) that react with amino groups on proteins or other molecules (Fig. 1). This kit contains all the reagents necessary for the labeling. The labeling process is very simple. Just add the NH2- reactive Biotin to IgG solution and incubate at 37oC for 10 minutes. 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 NH2-reactive biotin
♦ The molecular weight of the 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.
Easily Switch Fluorescence wavelength on your primary antibody
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Determination of Biotin/ Protein Ratio
The average number of biotin molecules per IgG molecule should be in the range of 5 and 8. If you need to determine the precise number of biotin molecules per Protein molecule use HABA assay. The following is a HABA assay protocol.
200 μM HABA (4-hydroxyazobenzene-2-carboxylic acid) solution prepared with PBS, pH 7.4 …………………… 1 ml
0.5 mg avidin/ml solution prepared with PBS, pH 7.4 ………………………………………………………………….. 1 ml
diluted sample solution (55 μl biotinylated protein solution + 110 μl PBS, pH 7.4)
25 μM biotin prepared with a mixed solution (2 volumes of PBS, pH 7.4 + 1 volume of WS buffer)……………… 200 μl
Prepare solutions of various concentrations (12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM) with serial dilution …………. 200 μl/ea
Fig. 2 Typical Calibration Curve of HABA Assay
1. Mix HABA solution and avidin solution in a plastic tube.
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, and 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 concentrations of biotin solution. 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 molecules per protein.