Protein Protocols Protein Protocols

Coupling of Abs with Biotin 355
48
Coupling of Antibodies with Biotin
Rosaria P. Haugland and Wendy W. You
1. Introduction
The avidin–biotin bond is the strongest known biological interaction between a
ligand and a protein (K d = 1.3 × 10 –15 M at pH 5) (1). The affinity is so high that the
avidin–biotin complex is extremely resistant to any type of denaturing agent (2). Biotin
(Fig. 1) is a small, hydrophobic molecule that functions as a coenzyme of carboxylases
(3). It is present in all living cells. Avidin is a tetrameric glycoprotein of 66,000–68,000
mol wt, found in egg albumin and in avian tissues. The interaction between avidin and
biotin occurs rapidly, and the stability of the complex has prompted its use for in situ
attachment of labels in a broad variety of applications, including immunoassays, DNA
hybridization (4–6), and localization of antigens in cells and tissues (7). Avidin has an
isoelectric point of 10.5. Because of its positively charged residues and its oligosaccharide
component, consisting mostly of mannose and glucosamine (8), avidin can interact
nonspecifically with negative charges on cell surfaces and nucleic acids, or with membrane
sugar receptors. At times, this causes background problems in histochemical and cytochemical
applications. Streptavidin, a near-neutral, biotin binding protein (9) isolated from
the culture medium of Streptomyces avidinii, is a tetrameric nonglycosylated analog of
avidin with a mol wt of about 60,000. Like avidin, each molecule of streptavidin binds
four molecules of biotin, with a similar dissociation constant. The two proteins have
about 33% sequence homology, and tryptophan residues seem to be involved in their
biotin binding sites (10,11). In general, streptavidin gives less background problems than
avidin. This protein, however, contains a tripeptide sequence Arg-Tyr-Asp (RYD) that
apparently mimics the binding sequence of fibronectin Arg-Gly-Asp (RGD), a universal
recognition domain of the extracellular matrix that specifically promotes cell adhesion.
Consequently, the streptavidin–cell-surface interaction causes high background
in certain applications (12).
As an alternative to both avidin and streptavidin, a chemically modified avidin,
NeutrAvidin (NeutrAvidin is a trademark of Pierce Chemical Company, Rockford,
IL) and its conjugates with enzymes or fluorescent probes are available from both
Molecular Probes and Pierce. NeutrAvidin consists of chemically deglycosylated avidin,
which has been modified to reduce the isoelectric point to a neutral value, without
loss of its biotin binding properties and without significant change in the lysines avail-
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
355