| NIH-3T3 embryonic cells grown to 85% confluency were harvested, homogenized in modified RIPA buffer (150 mM NaCl, 50 mM Tris-Cl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM EDTA, 1 mM PMSF, 5 µg/ml Pepstatin A, 2 µg/ml Leupeptin, 1 mM sodium orthovanadate, 1 mM sodium fluoride.), and cleared by centrifugation. Total protein concentration of the lysate was determined using BCA protein assay (BSA was used as a standard). Lysate was mixed with Laemmli Sample Buffer containing reducing reagent and was boiled for 5 minutes. Lysate is supplied as 200 µg in 100 µl per vial (2.0 mg/ml total protein concentration) in 1X Laemmli sample buffer. |
