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Fig. 5. Brain regions differ in gelatinolytic profi le. MMP-2 (68 and 62 kDa) and MMP-9 (92
and 84 kDa) signal from whole hippocampi (HIPPO) and the dentate molecular-layer (ML)
extracts following UEC lesion show similar patterns of lysis, but differences in signal
intensity. In fl uid percussion TBI, corpus callosum (CC) extracts reveal activity for both
gelatinases, but their relative contribution is altered compared with the hippocampal profi le.
l) containing 50 mM Tris buffer, pH 7.6, 150 mM
NaCl, 5 mM CaCl 2 , 0.05% Brij-35, 0.02% sodium azide, and
1% Triton X-100, centrifuged at 12,000 × g for 5 min at 4°C.
The supernatant is collected, aliquoted, and stored at −80°C
until assayed.
6. Gelatinases can be concentrated from culture media or tissue
extracts by using Gelatin-Sepharose 4B purifi cation methods
( 22, 23 ). This method takes advantage of targeted gelatinase
binding to the principal substrate gelatin, which is bound to
bead constructs for easy separation. Using one of these proto-
cols ( 22 ), we observed good zymographic signal and better
detection of the active form of MMP-2 in our brain tissue;
however, there may be problems in achieving consistent
removal of enzyme from the beads. We fi nd that multiple elution
steps are necessary to recover all bound gelatinases and we cannot
be certain that bound enzyme is equally eluted from both injured
and sham-injured control samples (see Fig. 2 ). It is possible
that the different cellular milieus generated by injured and
control conditions could alter Gelatin-Sepharose 4B binding
or elution from the beads. Further, interaction between gelatinases
and their endogenous binding partners may vary as a function
of injury condition and survival interval, making the applica-
tion of such concentration protocols more diffi cult to control.
For these reasons, we have focused our injury vs. sham-injured
control comparisons on whole-tissue extracts.
buffer (500
μ
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