Human Pulmonary Fibroblast-adult Lysate is prepared in modified RIPA buffer (50mMTris, pH7.2, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% NP-40, 1mM Na3VO4, 5mM NaF, 400µM PMSF, 1µg/ml of pepstatin A, 5µg/ml of aprotinin, 5µg/ml of leupeptin).
Cell debris is removed by centrifugation, and the protein concentration is determined with the Bio-Rad protein assay. The cell lysate is diluted and boiled for 5 min with nonreducing sample buffer (50 mM Tris-HCl, pH 6.8, 5% glycerol, 1% SDS, 0.002% bromphenol blue) containing 5% beta-mercaptoethanol.
Lysate quality was assured with a NuPAGE 4-12% Bis-Tris Gel stained with Coomassie brilliant blue.
For maximum recovery of product, centrifuge the original vial after thawing and before removing the cap. Aliquot to avoid repeated freezing and thawing.
Cell lysate need to to be boiled for 5 minutes before loading on SDS-PAGE for western blot. 40 μg per lane is recommended for mini gel.
Storage
Stable for 6 months at -20ºC from date of shipment.
Shipping
Dry-ice