Protein Protocols Protein Protocols

Avidin-or Streptavidin–Biotin 415
55
Utilization of Avidin or Streptavidin-Biotin as a Highly
Sensitive Method to Stain Total Protein on Membranes
Kenneth E. Santora, Stefanie A. Nelson, Kristi A. Lewis,
and William J. LaRochelle
1. Introduction
Since the initial publication by Towbin and coworkers (1) on the preparation of
replicas of sodium dodecyl sulfate (SDS)-polyacrylamide gel patterns, commonly
called protein blots, the technique of transferring proteins from inaccessible gel matrices
to accessible solid supports, such as nitrocellulose or nylon membranes, has become
widely utilized (2,3).
The detection of proteins on blots has ranged from the specific visualization of an
individual protein of interest to the general staining of total protein (4). Specific proteins
are detected with probes, such as antibodies or toxins, that are either directly radiolabeled
or conjugated to an enzyme (5,6). Alternatively, bound and unlabeled antibodies
or toxins are amplified by a secondary affinity probe similarly conjugated. The use of
biotinylated antibodies or toxins followed by avidin or streptavidin enzyme conjugates
is also gaining in popularity (7).
Total protein detection is usually based on a chemical affinity staining method such
as Coomassie Blue (8), Amido Black (9), India ink (10), oxidation/ derivation of carbohydrate
moieties (11,12), silver-enhanced copper detection (13) or Ponceau S (14). Other
approaches often require the chemical modification of the polypeptide with hapten followed
by detection with anti-hapten antibody and labeled secondary, protein A, or protein
G (15).
Here, we exploit the high-affinity and well-characterized interactions of biotin with
either avidin or streptavidin (16–18). Initially, proteins are resolved by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose
membrane, and the amino groups covalently derivatized (19) with sulfosuccinimidobiotin.
Depending on the sensitivity required, either of two techniques illustrated in Fig. 1 are
used to stain the proteins, which appear as dark bands against an essentially white
background (19,20). The first method utilizes avidin or streptavidin conjugated to
horseradish peroxidase (HRP) and detects