EZ Biotin Quantitation Kit From Pierce

Biotinylation of proteins is one of the most common formats for creating biomolecules for use as detection reagents. Because it is such a widely used technique, there are a large number of conjugation procedures and commercially available kits for the biotinylation of proteins. Since biotin is a much smaller molecule than the protein it is conjugated to, multiple biotins are coupled to a single protein. Quantifying the molar ratio of biotin to protein is valuable for measuring the success of the biotinylation process and subsequent function of the biotinylated proteins. The EZ Biotin Quantitation Kit from Pierce Biotechnology is used to measure the biotin levels in protein samples and thereby measure the molar ratio of biotin to protein in the sample.

HABA ([2-(4’-hydroxyazobenzene)] benzoic acid) and avidin are the basis of the kit. The measurement of biotin is achieved by displacement of HABA from the HABA:avidin complex; the HABA is displaced by the biotin in your sample. The displacement of HABA results in a change of absorbance measured at 500 nm. The absorbance change is directly proportional to the level of biotin in the sample. The kit contains all the reagents required for the quantitation and includes a generous aliquot of Pierce’s ImmunPure® Biotinylated Horseradish Peroxidase which is used as a standard. The HABA/Avidin Reaction Mixture is provided in Pierce No-Weigh™ individual tubes which provide just enough lyophilized reagent for a single reaction. The benefit of the single reaction tube is a reduction in the repetitive handling of the stock reagent that would occur if the reagent were provided in bulk. However, if your quantitation parameters require any modification of the kit protocol, the single use tubes will be difficult to work with.

I have primarily used the kit for the quantitation of biotin on antibodies. I found the kit protocol very simple, clear and easy to follow, and the procedure to be very quick. The kit will function regardless of the biotinylation technique as long as the sample solution lacks extremely high or low pH, organic solvents, unreacted biotin, detergents or other extremely harsh conditions. The only requirement is that the samples are in a PBS or TBS solution; the greater majority of protein samples will already be in these buffers and will not require dialyzation to be quantitated with the kit. I found the kit to be quite accurate. Even on the initial use of the kit, my standards measured exactly as expected, so I did not have to waste any reagent evaluating how well the kit functioned. The only problem I encountered with the kit was with protein samples with lower concentration levels (approximately 100 µg/mL). These samples had such small amounts of biotin that they were barely detected. However, with a small amount of freelancing with the kit protocol volumes, this can be addressed and the kit still functions well. For some samples containing around 100 µg of material, I used a smaller reaction volume and Pierce Slide-A-Lyzer Mini Dialysis Units instead of the Pierce Slide-A-Lyzer Cassettes with good results. The calculations required to determine the biotin molar ratios are simple and covered thoroughly in the protocol, with examples provided. The kit is also compatible with a microplate or cuvette spectrophotometer formats.

The price of the kit is reasonable. A single kit contains 24 of the No-Weigh™ HABA/Avidin Premix tubes. With running a few standards, this is sufficient for a little more than 20 samples. The kit is extremely easy to use, robust and accurate. There is really no reason to create a homebrew to perform this measurement because the kit that is so easy to use and relatively cheap. The time spent formulating the reagents would not offset the expense of just purchasing the kit.