Precaution:♦ 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 RatioThe 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.
Reagent solution:200 μM HABA (4-hydroxyazobenzene-2-carboxylic acid) solution prepared with PBS, pH 7.4 …………………… 1 ml0.5 mg avidin/ml solution prepared with PBS, pH 7.4 ………………………………………………………………….. 1 mldiluted 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 μlPrepare 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.
是的，如果分子量大于 50,000。在标记蛋白质之前是否必须使用过滤管？
如果蛋白溶液不含带氨基的小分子，蛋白浓度为10 mg/ml或约70 μM，则无需使用过滤管。只需将 10 μl 样品溶液与 90 μl 反应缓冲液混合，然后将混合物添加到一瓶 NH2 反应性生物素中。反应后，将所有反应混合物转移到过滤管中，然后按照从步骤 6 开始的方案进行操作。
我必须使用 WS 缓冲液来储存生物素标记的蛋白质吗？
您不必使用 WS 缓冲区。您可以根据您的实验选择任何类型的缓冲液。
我的样品含有少量不溶性物质。我该怎么办？
旋转样品并使用上清液进行标记。
生物素标记的蛋白质能稳定多久？
如果您将生物素标记的蛋白质储存在 0-5ºC，它可以稳定保存 2 个月。如需更长的储存时间，加入 100% 体积的甘油，等分，并储存在 -20ºC（如果蛋白质可以冷冻）。但是，请注意稳定性取决于蛋白质本身。
该试剂盒可以标记的最少 IgG 量是多少？
最低量为 10 μg IgG；只需遵循协议。 10 μg 至 100 μg IgG 的标记比率保持不变。