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Fig. 2. Effects of injury and tissue extraction methods on gelatinolytic activity. Detergent-
extracted (T-PER) hippocampal tissue from different injury models shows increases in
both MMP-2 and MMP-9 activity as compared to the sham control. Comigration of sample
pro-MMP-9 with pro-MMP-9 standard (92 kDa) is shown in center panel. Extraction of
MMP with Brij/Triton X-100 followed by MMP concentration with Gelatin-Sepharose 4B
and bead elutions (E1 and E2) result in an increased signal for MMP-2 active form (62 kDa)
as shown in
right panels.
Corpus callosum and subregional cerebral cortex samples are
extracted in RIPA Lysis Buffer (working solution: 50 mM Tris-
HCl, pH 7.4, 0.15 M NaCl, 0.25% deoxycholic acid, 1%NP-40,
1 mM EDTA), in volumes of 125 and 300
l, respectively. These
samples are also homogenized on ice for 30 s using polypropylene
microcentrifuge tubes and corresponding motorized pestles. The
RIPA homogenate is then centrifuged at 14,000 ×
g
for 20 min, at
4°C. RIPA supernatant is collected, aliquoted, and stored at −80°C
until assayed for protein using Pierce BCA Protein Assay Kit and
subsequent zymography analysis. As for hippocampus, we fi nd these
samples to be stable for up to 6-8 months when stored at −80°C.
μ
3.2. Gel
Electrophoresis
and Incubation
Proteins from T-PER and RIPA extracts are identically separated
by SDS-PAGE prior to zymographic detection of gelatin proteolysis
using the Zymogram Gelatin Gel System from Invitrogen (Note 7).
Aliquots containing 20
g protein are removed from stored T-PER
or RIPA supernatant, mixed with unreduced Tris-Glycine SDS
sample buffer, and allowed to sit for 10 min at RT. This concentra-
tion of protein works well, avoiding the artifacts of protein overload
(Fig.
1
; Note 8). Proteins are electrophoresed on a 10% Zymogram
Gelatin Gel in Tris-Glycine SDS running buffer, at 4°C and 125 V
for 1.5 h. Control samples are run on each gel when experiments
require multiple zymograms (Note 9). Gels are then transferred to
a plastic dish with a tight lid and incubated in 100 ml Renaturing
μ
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