1- Rinse the cells twice with ice-cold phosphate-buffered saline (PBS).
2- Harvest the cell pellet in ice-cold PBS supplemented with protease inhibitors and sonicate. Alternatively, solubilize the cells in the presence of 1% NP-40, a mild detergent diluted in PBS, for 20 min at 4°C under agitation and centrifuged at 16,000 × g for 10 min to remove cellular debris.
3- Measure the protein content by a small-volume micromethod using the Bio-Rad DC protein assay.
4- Mix 30-40 µg of lysates at 2 µg/µl of protein with doubly concentrated PFO sample buffer (100 mM Tris base, 2-8% NaPFO (Oakwood Products Inc), 20% glycerol, and 0.005% bromophenol blue, pH adjusted at 8.0 with NaOH) and 25 mM DTT.
5- Incubate at room temperature for 25 min, vortex briefly, and centrifuge 5 min at 10,000 × g.
6- Load your samples at room temperature on a 4-12% precast gradient Novex Tris-Glycine gel without SDS (Invitrogen) along with molecular weight standards resuspended in PFO sample buffer (high-molecular-mass rainbow marker kit (GE Healthcare), cross-linked Albumin and phosphorylase b (Sigma)).
7- Perform electrophoresis at 140 V with a running buffer (25 mM Tris, 192 mM glycine, 0.5% NaPFO, pH 8.5, adjusted with NaOH) precooled to 4°C. During the run, the electrophoresis box is kept at 4°C.
8- Transfer the proteins to a nitrocellulose membrane for 1 hr in a transfer buffer containing 25 mM Tris, 192 mM glycine and 20% (v/v) methanol.
9- Analyze the protein band sizes either by Amido black staining (Sigma) for the molecular weight standards or by standard immunoblotting.