Sciatic Nerve Protein Extraction

P10 Adam23^Δ1/Δ1 and age-matched WT control mice were culled, and sciatic nerves were harvested into Eppendorf tubes, snap-frozen in liquid nitrogen, and stored at −80°C until further processing. Nerves were homogenized using a Sample Grinding Kit and homogenization buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM Na₃VO₄, 2.5 mM Na₄P₂O₇, 20 mM NaF, 2.5 mM β-Glycerophosphate, 1% NP40, 1% Tx100, 1% SDS, 1% Deoxycholate, PMSF, PIM). Samples were then centrifuged at 4°C for 10 min at 20,000 × g, and supernatants were transferred into fresh tubes. Protein concentration was measured using the Pierce Protein Assay (BCA). Samples of appropriate, equalized protein concentrations and volumes were mixed with LDS Sample Buffer and frozen at −20°C or immediately used in WB.