Protein Protocols Protein Protocols

418 Santora et al.
7. After allowing sufficient time for color development (usually 30 min), rinse the replicas
with distilled water, and dry between two sheets of dialysis membrane. The replicas may
also be dried, and stored in cellophane for future use.
3.2. Streptavidin/Antistreptavidin Amplified Staining
1. If desired, an alternative procedure to amplify fivefold the detection method described
above is utilized. First, biotinylate and block the nitrocellulose filters as described in Subheading
3.1., steps 1–4 of the avidin- or streptavidin-staining protocol.
2. Incubate blots with streptavidin (1 µg/mL) for 1 h in PBS containing 1% BSA, 0.05%
Tween-80. Wash the replicas three times for 15 min each time with wash solution.
3. Next, dilute affinity-purified rabbit antistreptavidin IgG (0.5 µg/mL) in PBS containing
1% BSA, 0.05% Tween-80. Add solution to replica for 4 h or overnight if convenient.
Wash replicas three times for 15 min each time with wash solution.
4. Incubate replicas with goat antirabbit immunoglobulin conjugated to horseradish peroxidase
diluted (4 µg/mL) in PBS containing 1% BSA, 0.05% Tween-80 for 4 h. Wash replicas
three times for 15 min each time with wash solution.
5. Protein bands are visualized by immersing the replicas in α-chloronaphthol solution for
approx 30 min (see Note 6). The replicas are rinsed with distilled water and dried between
two sheets of dialysis membrane.
4. Notes
1. This method is more sensitive for nitrocellulose membranes than for proteins transferred
to Biodyne membranes by approx 10-fold owing to the higher background staining of
Biodyne membranes.
2. Because of the sensitivity of the staining, care should be taken to avoid protein contamination
of replicas with fingertips, and so forth. Gloves or forceps should be used.
3. This method derivatizes the free amino groups of proteins bound to filters. Amine containing
compounds, such as Tris or glycine buffers, will compete for biotinylation with
the sulfosuccinimidobiotin. Blot transfer buffers that contain free amino groups, such as
Tris or glycine, may be used, but must be thoroughly removed by soaking and rinsing as
indicated in Subheading 3.1., step 1.
4. The staining is highly dependent on the sulfosuccinimidobiotin concentration. Sulfosuccinimidobiotin
concentrations of >10 µM have resulted in a dramatic decrease of protein
staining intensity (12). In some instances, it may be necessary to determine empirically the
optimal concentration of sulfosuccinimidobiotin to use for staining particular proteins.
5. Little or no differences were observed when avidin conjugated to horseradish peroxidase
was substituted for streptavidin conjugated to horseradish peroxidase. However, for some
applications, streptavidin may present fewer problems owing to a neutral isoelectric point
and apparent lack of glycosylation.
6. Sodium azide will inhibit horseradish peroxidase and, accordingly, must be removed
before addition of the enzyme solution.
7. All reagent concentrations were determined empirically and were chosen to give maximum
staining sensitivity. In principle, our procedure can be used in double-label experiments
in which all proteins on the replica are biotinylated and the same blot is then probed
with radioactive antibody or protein A.
Acknowledgments
The author thanks Stanley C. Froehner for helpful advice, discussions, and continued
encouragement. This work was supported by grants to SCF from the NIH (NS-14781)
and the Muscular Dystrophy Association.