Tips for Biotin, Avidin, & Streptavidin


The streptavidin-biotin system is a protein-ligand interaction present in nature that has been successfully used in a number of applications including detection of proteins, nucleic acids, and lipids as well as protein purification. The avidin-biotin system is a simple yet elegant system to link proteins in immunoassays by exploiting the very high affinity of hen egg-white avidin for biotin (vitamin B7). Streptavidin, isolated from bacteria, binds to biotin equally well but lacks the glycoprotein portion found on avidin and therefore shows less non-specific binding. In immunoassays, antibodies and reporters like fluorochromes or enzymes that are used to localize or quantitate analytes are often coupled via a streptavidin/avidin-biotin bridge. Here, you will find some useful tips to keep in mind when working with the streptavidin-biotin affinity system.

  • Avidin or Streptavidin?

    Although both avidin and streptavidin bind to biotin with very high affinity, the major problem of using avidin in some applications is the high non-specific binding, which is attributed to both the presence of the sugars and high pI. Thereby, significant non-specific binding can be prevented with the use of streptavidin or alternatively, deglycosylated avidins that still preserve the same biotin-binding properties.

  • Streptavidin-biotin as a versatile detection system

    The streptavidin-biotin system can be incorporated into virtually every immunoassay, whereby an antibody is conjugated to biotin and then detected with avidin or streptavidin. It is then conjugated to inexpensive, high-quality variety of fluorochromes and enzymes, widely available from commercial sources. This makes biotinylated antibodies advantageous to signal amplification and increased sensitivity, but at the same time, demands optimization of antibody and conjugate dilutions.

  • Choose the right buffer system when working with biotinylated antibodies

    In order to prevent high background and low signal-to-noise ratios, it is highly recommended to avoid fetal bovine serum (FBS) or biotin in blocking buffers and other solutions. A good substitute would be 0.1%–2.0% BSA fraction V. In Western blots, nonfat dry milk or casein should be limited to the initial blocking step because of residual biotin that will interfere with the assay. In this case, antibody solutions should then be prepared in TBS-Tween, which is compatible with both nitrocellulose and PVDF membranes. If background is still a problem, highly purified 0.2%–6% casein may improve the results.

  • Reducing interference from residual biotin in samples

    Sometimes non-specific bands might be observed by Western blot when using the streptavidin-biotin system. This is especially true for tissue preparations and cell lysates since they might contain enzymes with covalently bound biotin as a cofactor. In these cases, it is useful to increase the ionic strength of buffers (~0.5 M NaCl) and/or perform specific biotin blocking step before incubation with the primary antibody. In this instance, removal of free biotin and biotinylated enzymes from casein by using streptavidin agarose beads can significantly enhance sensitivity.

  • Biotinylation of cell surface for protein quantification

    The streptavidin-biotin system can be successfully used in a noninvasive technique to chemically tag cell-surface proteins for direct, accurate quantitation of cell-surface expression, endocytosis, and recycling of a variety of plasma-membrane receptors and antigens. The amount of biotin bound to a specific receptor or antigen can be subsequently quantitated using ELISA or Western blot. This technique constitutes a rapid and safe alternative to radioactive labeling.

  • Protein biotinylation

    When preparing a reagent for biotin-labeling of proteins it is advisable to conduct optimization assays under the system of study. This is because the choice of an appropriate spacer and the right chemistry to bind biotin to the target protein(s) may have a profound impact on the outcome of the experiment. The most widespread approach for protein biotinylation is based on modification of exposed primary amino groups in proteins with a succinimidyl (NHS) ester of biotin. When modification of free amino groups is not possible, proteins can still be biotinylated if they contain free thiol groups or if they are glycosylated. Moreover, it is also possible to biotinylate proteins using a commercially available photoreactive biotinylation reagent that activates after exposure to ultraviolet light.

  • Purification of biotinylated proteins

    Since streptavidin-biotin complexes require strong denaturing buffers to break their interaction and release the biotin, purification of biotinylated proteins from immobilized streptavidin would also result in loss of native configuration and functional properties. For this purpose, consider using avidin analogs designed to bind biotin with lower affinity and allowing elution to be carried out under milder conditions. Alternatively, 2-iminobiotin the cyclic guanidino analog of biotin could be used since it exhibits a pH-dependent interaction with streptavidin that facilitates recovery under milder conditions as well. It retains high-affinity specific binding at high pH but interacts poorly at acidic pH values. Alternatively, biotin with cleavable spacers can be also used.

  • Biotinylation including cleavable spacers

    The use of cleavable spacers between the bicyclic ring of biotin and the chemical group responsible for carrying out the covalent binding to proteins have been introduced with the goal of facilitating the release of biotinylated proteins after capture on immobilized streptavidin. These include disulfide bridges that can be broken by reducing agents like 2-mercaptoethanol or dithiothreitol and photocleavable linkers, which are readily available from commercial sources. Each of these approaches, however, presents its own limitations and should be carefully optimized to particular experimental conditions.