Immediately before electrophoresis, 3.5 μl of sample buffer (4×
NuPage sample buffer, Novex, San Diego, CA) containing dithiothreitol
(final concentration, 50 mM) was added to 6.5 μl of the protein
extract. The 4× sample buffer (pH 8.5) consisted of 1.17 M sucrose,
563 mM Tris base, 423 mM Tris-HCl, 278 mM sodium dodecyl sulfate (SDS),
2.05 mM EDTA, 0.88 mM Coomassie Blue R250 and 0.70 mM phenol red. After
heating for 10 minutes at 70°C, 10-μl volumes of each sample were
loaded into gel slots for electrophoresis on 10% gels (Bis-Tris;
Novex) with 2-(N-morpholino) ethane sulfonic acid SDS
running buffer (NuPage MES-SDS; Novex). Recombinant human TIMP-3
protein (10 ng) was included in a separate lane as a positive control.
Proteins were transferred from the gel to polyvinylidene difluoride
membranes using blotting apparatus (XCell II; Novex). After transfer,
membranes were incubated with blocking solution (2% BSA in
Tris-buffered saline) for 1 hour. The monoclonal anti-human TIMP-3
antibody (Clone 136-17B12) conjugated to horseradish peroxidase was
applied at 1:1000 dilution and incubated overnight at 4°C. After
rinsing, immunoreactivity was displayed with the chemiluminescent
method (ECL, Amersham, Arlington Heights, IL) and captured on
radiographic film during a 10- to 15-second exposure. The
immunoreactivity signal was digitized on a Scanwizard
(Microtek, Redondo Beach, CA) flatbed scanner. The intensity of
immunoreactivity was quantitated from the digitized images using
commercial software (Digital Science 1D; Eastman Kodak, Rochester, NY)
on a personal computer (Power Macintosh, Apple Computer, Cupertino,
CA). The densitometric intensity was converted to amount of protein
(nanograms per sample) by comparing the intensity of immunoreactivity
with that of varying amounts of recombinant TIMP-3 standard.