Biotinylation Protocols

Biotinylation is the process of linking biotin, or vitamin H, to a protein or other molecules. This scientific procedure is mostly used in biotechnology and molecular biology research involving DNA , proteins and antigens, which are molecules the body's immune system recognize as invaders. Biotinylation protocols include the biotinylation of antibodies and peptides, as well as cell surface biotinylation.

Biotinylation of Antibodies

• To link biotin to antibodies proteins produce when an antigen enters the body, technicians use sodium bicarbonate (NaHC03) at pH 9; dimethyl sulfoxide (DMSO);

  NHS-Biotin and a saline substance called PBS. Samples are mixed with sodium bicarbonate, which is later added to NHS-Biotin in DMSO. The solution is then added to the analyzed antibodies and incubated on a piece of equipment called a rotator for four hours at room temperature or overnight at 39.2 degrees Fahrenheit. Finally, the solution is diluted with PBS.

Cell Surface Biotinylation

• In the cell surface biotinylation protocol, technicians use tissue culture, phosphate-buffered saline (PBS) at pH 7.4 and sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate buffer solution to balance acidity containing amino groups and TrisCl, according to Current Protocols in Protein Science.The cells are mixed with the biotin and buffer solution, decanted and centrifuged. Solid contents and specific proteins are later analyzed on a gel electrophoresis plate.

Biotinylation of Peptides

• To perform the biotinylation of peptides, the reagent used is NHS-SS Biotin, according to Philip Shepherd in "Monoclonal Antibodies: A Practical Approach." Peptides are short fragments of amino acids, the molecules that form proteins and DNA. In this protocol, the commercial solution NHS-SS Biotin is dissolved in water. Then the peptide sample is added to it, together with sodium bicarbonate to balance acidity or pH. The solution is then incubated on ice for two hours. Technicians can observe the sample with or without the biotin molecules.