Biotin Chromogenic Detection Kit From Fermentas

The Biotin Chromogenic Detection Kit by Fermentas was a developed for the detection of biotinylated nucleic acid fragments through the manipulation of the streptavidin-biotin interaction system. Streptavidin is a protein found in the bacteria Streptomyces avidinii which forms a very strong bond with biotin. It is the strongest noncovalent biological interaction documented to date.

I use this detection system to detect DNA probes that have been labeled with biotin using the Biotin DecaLabel kit by Fermentas, after probing positively charged nylon membranes during Southern blotting.

The kit operates on a two-step principle. In the first step, biotin that has been incorporated into the DNA probe binds to the streptavidin provided with the Biotin Chromogenic Detection Kit. In the second step, alkaline phosphatase, which is conjugated to the streptavidin, cleaves a substrate to generate a colored precipitate which enables detection of specific DNA bands. Although I only use this kit for Southern blotting, the principle can be applied for the detection of labeled nucleic acids in a number of different applications.

The substrate which is cleaved by alkaline phosphatase is BCIP-T. It forms an insoluble blue precipitate after cleavage and this can be detected visually without the requirement of any equipment that enhances colorimetric detection.

One thing I like about this kit is that it provides a robust supplementary protocol for hybridization, which we implemented during our Southern blotting procedure. The protocol itself is a fairly standard hybridization method, but it left us with the peace of mind that the conditions for optimum biotin binding and detection were achieved while using the kit.

The manufacturer’s state that the detection limit of a target sequence is as low as 50 fg. This is clearly very appealing when working with samples with notoriously difficult DNA extraction or problems with probe design. Another recommendation by the manufacturers is that this kit should not be used for in situ hybridization. This is worth keeping in mind because the biotin labelling kit provided by the same manufacturer’s works perfectly well for in situ hybridizations, therefore alternative methods need to be explored for this specific application.

The protocol itself is designed for a membrane of 100 cm². Volumes and concentrations, therefore, need to be adjusted depending on the size of the membrane for any given experiment. Also, most solutions provided in the kit need to be diluted and adjusted just prior to use. The method then is a series of washes and incubations which take a total of about 3 to 4 hours. After this, it is possible to observe the blue precipitate which is indicative of your target nucleic acid fragment in just 30 minutes after the addition of the substrate. All in all, this is an elegant kit that utilizes a very powerful biological interaction to enable highly sensitive chromogenic detection.