The high affinity of avidin for biotin was first exploited in histochemical applications in the mid-1970s. This egg-white protein and its bacterial counterpart, streptavidin, have since become standard reagents for diverse detection schemes. In their simplest form, such avidin–biotin detection methods entail applying a biotinylated probe to a sample and then detecting the bound probe with a labeled avidin or streptavidin. These techniques are commonly used to localize antigens in cells and tissues and to detect biomolecules in immunoassays and DNA hybridization procedures. In some applications, immobilized avidins are used to capture and release biotinylated targets. In addition to our dye and enzyme conjugates of avidins and streptavidins, this section contains several products that can be used for the affinity isolation of biotin-conjugated molecules and their complexes in cell and tissues.
Our diverse set of biotinylation reagents and biotin conjugates are described in Biotin and Desthiobiotin Conjugates—Section 4.3. Combining one of our biotinylated or DSB-X biotin–labeled secondary antibodies (Secondary Immunoreagents—Section 7.2, Summary of Molecular Probes secondary antibody conjugates—Table 7.1) with a fluorescent dye– or enzyme-labeled avidin provides an sensitive method for the indirect detection of antibodies from various animal sources.
Avidin, streptavidin and Thermo Scientific™ NeutrAvidin™ biotin-binding protein each bind four biotins per molecule with high affinity and selectivity. Dissociation of biotin from streptavidin is reported to be about 30 times faster than dissociation of biotin from avidin. Their multiple binding sites permit a number of techniques in which unlabeled avidin, streptavidin or NeutrAvidin biotin-binding protein can be used to bridge two biotinylated reagents. This bridging method, which is commonly used to link a biotinylated probe to a biotinylated enzyme in enzyme-linked immunohistochemical applications, often eliminates the background problems that can occur when using direct avidin– or streptavidin–enzyme conjugates. However, endogenously biotinylated proteins that have carboxylase activity are found in the mitochondria; therefore, sensitive detection of biotinylated targets in cells requires the use of biotin-blocking agents to reduce this background. Our Endogenous Biotin-Blocking Kit provides the reagents and a protocol for this application. Nonspecific binding of avidin conjugates of enzymes to nitrocellulose can be blocked more effectively by adding extra salts to buffers rather than by adding protein-based blocking reagents.
High-purity unlabeled avidin, streptavidin, NeutrAvidin biotin-binding protein and Invitrogen™ CaptAvidin™ biotin-binding protein are available in bulk. We also offer avidin specially packaged in a smaller unit size for extra convenience. Our avidin, streptavidin and deglycosylated NeutrAvidin biotin-binding protein typically bind greater than 12 µg of biotin per mg protein.
Avidin is a highly cationic 66,000-dalton glycoprotein with an isoelectric point of about 10.5. It is thought that avidin's positively charged residues and its oligosaccharide component (heterogeneous structures composed largely of mannose and N-acetylglucosamine) can interact nonspecifically with negatively charged cell surfaces and nucleic acids, sometimes causing background problems in some histochemical applications and flow cytometry. Methods have been developed to suppress this nonspecific avidin binding. In some cases, avidin's nonspecific binding can also be exploited. For example, avidin and its conjugates selectively bind to a component in rodent and human mast cell granules in fixed-cell preparations and can be used to identify mast cells in normal and diseased human tissue without requiring a biotinylated probe.
Streptavidin, a nonglycosylated 52,800-dalton protein with a near-neutral isoelectric point, reportedly exhibits less nonspecific binding than avidin. However, streptavidin contains the tripeptide sequence Arg–Tyr–Asp (RYD) that apparently mimics the Arg–Gly–Asp (RGD) binding sequence of fibronectin, a component of the extracellular matrix that specifically promotes cellular adhesion. This universal recognition sequence binds integrins and related cell-surface molecules. Background problems sometimes associated with streptavidin may be attributable to this tripeptide. We have particularly observed binding of streptavidin and anti-biotin conjugates to mitochondria in some cells that can be blocked with the reagents in our Endogenous Biotin-Blocking Kit.
We provide an alternative to the commonly used avidin and streptavidin. Our conjugates of NeutrAvidin biotin-binding protein—a protein that has been processed to remove the carbohydrate and lower its isoelectric point—can sometimes reduce background staining. The methods used to deglycosylate the avidin
