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Posted on Thu, Aug 10, 2017

Biotinylated proteins and oligonucleotides

Biotinylation is the process of attaching a biotin tag to a molecule. The molecule can be a protein or an oligonucleotide. Biotinylated molecules are useful in many biological contexts, but are primarily used for capture or detection of target molecules. Biotinylated molecules are used in Western blotting, ELISA, flow cytometry, and other inventive detection methods.

Why Biotin?

Biotin is one half of a famous natural affinity pair. The other half is streptavidin. The two proteins experience highly specific non-covalent binding with each other. The affinity is resistant to changes in temperature or pH, and it can remain intact even in the presence of some organic solvents and denaturing agents. The highly specific nature of the affinity has made the streptavidin-biotin pair a standard tool in modern biotechnology.

Biotin itself is a water-soluble B vitamin. Its small molecular weight of 244.31 g/mol makes it unlikely to interfere with the function of the larger protein it is attached to. Biotinylation can be accomplished with a general chemical reaction or with a more specific enzymatic reaction. 

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Biotinylation 

Biotinylation is the process of attaching a biotin tag to a molecule such as an antibody, magnetic nanoparticle, or DNA probe. Biotin has a long hydrocarbon side chain with a carboxyl group at the end. This valeric acid side chain chemically reacts with amines or thiols. Biotin tags are often covalently attached to lysine amino acids on antibodies and other globular proteins. In this way many biotin molecules can be attached to a single molecule. This chemical reaction method is quite general, and doesn't provide much control over the location of the biotin tag on the protein. Enzymatic biotinylation techniques are available to improve control over the location of the biotin tag. These enzymatic techniques rely on tags and specific enzymes to target a particular location on a protein. Commercial kits are available to simplify and streamline the chemical or enzymatic biotinylation process.  

Biotin-modified thymidine (Biotin dT) residues are incorporated into the oligonucleotide to produce Biotinylated oligonucleotides.  Another modification called Biotin-TEG is useful for adding biotin tags to magnetic nanoparticles. This biotin modification has a long triethyleneglcol (TEG) spacer between the nucleic residue and the biotin molecule in order to reduce steric hindrance between biotin groups. There are many other biotin modifications commercially available. Some of these include modifications that allow post-binding release of a biotin analog from streptavidin.

 

Biotinylation and magnetic beads

Biotinylated antibodies or oligonucleotides are often immobilized on a solid surface. Magnetic beads are particularly useful for this application because of their high surface area and recoverability. One recently published application involves a DNA oligonucleotide probe containing endonuclease restriction sites and a biotin tag at the end. The probe is immobilized on magnetic beads and incubated in a solution containing DNA methyltranferase, which methylates the DNA and protects it from restriction by the endonuclease. After incubation the magnetic beads are collected by magnetic separation and incubated with streptavidin-conjugated alkaline phosphatase to produce a readout of DNA methylation efficacy. If the methyltransferase methylated the DNA probe, then the endonuclease couldn't cleave the DNA, and the biotin tag remains attached, which then binds to the streptavidin alkaline phosphatase, which then produces a signal. If the methyltransferase is not present, then the probe is cleaved and the signal diminishes.

 

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