Friday September 12, 2008
Conjugation of a biotin moiety to a protein or peptide is arguably the easiest method by which to attach a handle for later affinity capture or other biochemical assay. While most commercial biotinylation kits are relatively straightforward, they frequently require extensive hands-on time. In addition, most kit protocols include a step to remove unreacted biotin which tends to protein loss. Innova Biosciences (Cambridge, UK) has developed a biotinylation kit, called Lightning-Link™, that is hands down the simplest kit on the market. The entire Lightning-Link biotinylation protocol is carried out in a single microcentrifuge tube with a total hands-on time of less than one minute.
The basic protocol is to add 1 µl of Lightning-Link modifier (LL-modifier) reagent for every 10 µl protein solution. The total amount of protein depends on which kit you are using and ranges from low µg to 2 mg. The instructions also specify optimal volumes of protein solution. The protein/modifier mixture is then added to a tube of lyophilized reagent mixture and allowed to react for 3 h to overnight. The reaction is stopped by the addition of LL-quencher reagent (1 µl per 10 µl protein solution) followed by a further 30 min incubation. The biotinylated protein is ready for immediate use. Since there is no need for removal of unreacted biotin, protein recovery is 100%.
We performed a Lightning-Link biotinylation of 100 µg of a purified protein and tested the resulting conjugate by capturing the protein, with or without biotin, on a streptavidin-coated ELISA plate. Detection of bound biotinylated protein with streptavidin-HRP showed that the protein had indeed been biotinylated in at least 2 positions per protein molecule (i.e. the protein was captured via a biotin moiety and detected via a different biotin moiety on the same protein). We also determined that the biotinylation of the protein did not alter its recognition by a mAb against the protein.
Another advantage of the Lightning-Link system is that the conjugation reaction can be carried out at or near physiological pH (pH range 6.5-8.5), eliminating the possibility of protein damage due to pH extremes. Although amine-free buffers are recommended, the conjugation reaction is not affected by low concentrations of Tris. Lightning-Link Biotin Conjugation kits are available in two sizes, for up to 200 µg or 2 mg protein. Each of these sizes is available in two flavors, one designed for capture of the biotinylated protein on a streptavidin-coated plate and the other for detection with a streptavidin-conjugated reagent (e.g. streptavidin-HRP). Lightning-Link Biotin Conjugation kits are distributed by Novus Biologicals (Littleton, CO) in the US.
Michael Campa, PhD
Associate Research Professor
Department of Radiology
Duke University Medical Center
United States