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Fig. 1. Effects of protein load and hemorrhage on hippocampal gelatin zymography. High
concentrations of protein per lane (100
μ
g) interfere with visualization of lytic bands (
a
). Use
of samples from TBI + BEC cases with extensive hemorrhage shows signifi cant increase
in overall lytic activity (
right lane
in
b
) when compared to the typical profi le for MMP-2 and
MMP-9 (
left lane
) .
fi rst to gain better access for dissection. Rinse the dissected tissue
with sterile saline before extraction to remove any excess surface
blood (Fig.
1
; Notes 2 and 3). Fresh tissue is then homogenized
directly in extraction buffer (for our samples, T-PER, RIPA, or Brij).
We routinely dissect hippocampus, cerebral cortex, and corpus
callosum from cases subjected to unilateral entorhinal cortical
lesion (UEC), moderate central fl uid percussion TBI, or combined
TBI and bilateral UEC (TBI + BEC), as well as paired sham-injured
animals. This method permits successful extraction of tissue gelati-
nases using a variety of buffer systems, both with and without
detergent (Notes 4 and 5; see also Table
1
. for details of published
extraction buffers).
Hippocampi are routinely homogenized in 250
l T-PER extraction
buffer which uses a proprietary detergent in 25 mM Bicine and
150 mM NaCl, pH 7.6. Each brain sample is homogenized on ice
for 30 s in polypropylene microcentrifuge tubes using a cordless
motor homogenizer. The T-PER homogenate is then centrifuged
at 8,000 ×
g
for 5 min at 4°C. The T-PER supernatant is collected,
aliquoted for single use, and stored at −80°C until assayed for
protein (Pierce BCA Protein Assay) and used for zymography. In
some protocols, further processing of samples for MMP concen-
tration using Gelatin-Sepharose 4B may be employed (see again
Fig.
2
, Note 6; also Table
1
). Samples are stable and may be stored
for up to 6-8 months at −80°C.
μ
3.1.2. Protein Extraction
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