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Frozen spinal cord ~50 mg was homogenized in 05 mL of ice cold methanol containing 100 pg/mL of internal standard using TissueLyserLT (Qiagen) for 2 × 5 minutes at 50 Hz Another 05 mL of methanol was added and samples sonicated using probe sonicator at 30% until no course tissue remained
A precooled TissueLyserLT (QIAGEN) was used to homogenize tissue samples in a buffer containing 20 mM TrisHCl (pH 75), 1% Triton X100, 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1% sodium deoxycholate, 25 mM sodium pyrophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor cocktails (Roche Diagnostics) The protein
The tissue was mechanically disrupted using a bead mill homogenizer (TissueLyserLT; Qiagen) for 5 minutes at least twice until tissue was completely homogenized and centrifuged at 15,000g for 15 minutes The protein fraction of the centrifuged sample was extracted, aliquoted and stored at −80 °C until further use The protein content of the
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