Biotin, a 244Da vitamin (Vitamin H) molecule, exhibits an extraordinary binding affinity for avidin (K_a=10¹⁵ M^-1) and streptavidin (K_a=10¹⁵ M^-1). Biotin and (strept)avidin interaction is rapid and once the bond is established it can survive up to 3M guanidine-hydrochloride and extremes of pH. Biotin-avidin bonds can only be reversed by denaturing the avidin protein molecule with 8M guanidine-hydrochloride at pH1.5 or by autoclaving. The biotinylated molecules are efficiently probed with avidin or streptavidin conjugated to reporter molecules, such as peroxidases or phosphatases. The use of biotin for non-radioactive labeling of proteins and nucleic acids has now become an increasingly popular technique in life science research. The use of biotin conjugation has eliminated the hazards, instability, and disposal problems associated with the use of radioisotopes.

 

Avidin is a glycoprotein with approximately 10% of its total mass coming from carbohydrates. Avidin has a molecular weight of 67kDa and contains four identical 128 amino acid subunits that each have a single biotin binding domain. Avidin is a basic protein with an isoelectric pH of 10-10.5 and is readily soluble in aqueous buffers containing a wide range of salt, pH, temperature and other laboratory agents. This wide range of tolerance makes avidin suitable for a wide variety of analytical applications.

 

Streptavidin is a tetrameric protein and in many respects is similar to avidin except that it has no carbohydrate and has a slightly lower molecular weight of about 60kDa. The solubility of streptavidin (isoelectric pH5) in aqueous buffer is much lower than avidin, but the binding of streptavidin to biotin is similar to that of avidin. The advantage of streptavidin is that the lack of carbohydrates significantly reduces the amount of non-specific binding.

 

The Steptavidin Resin is designed for the single step affinity purification of proteins and antibodies with a biotin tag.  The resin consists of steptavidin coupled to ~4% cross-linked agarose, via a 15 carbon spacer arm.