Ammonium persulfate

Soft gel mixes contained: 550 µl of 7.6 mM hydrochloric acid (HCL), 330.5 µl of double-distilled water (ddH₂O), 0.5 µl N,N,N′,N′-tetramethylethylenediamine (TEMED) (Sigma), 20 µl 2% bis-acrylamide (BioRad), 70 µl of 40% acrylamide (BioRad), 20 µl 0.1 M NHS (N-hydroxysuccinimide, Sigma-Aldrich), 4 µl of 200 nm diameter beads resuspended at 0.2 µM (Invitrogen) and 5 µl of 10% ammonium persulfate (GE HealthCare) (prepared just before use). Stiff gels mixes contained: 550 µl of 7.6 mM HCL, 258.5 µl of ddH₂O, 0.5 µl of TEMED (Sigma), 25 µl 2% bis-acrylamide (BioRad), 137 µl of 40% acrylamide (BioRad), 20 µl of 0.1 M NHS (N-hydroxysuccinimide, Sigma-Aldrich), 4 µl of 200 nm diameter beads resuspended at 0.2 µM (Invitrogen) and 5 µl of 10% ammonium persulfate (GE HealthCare) (added just before use). A 12-μl drop of PAA mix was placed into the hydrophilic glass of a glass bottom dish (FD5040-100). The PAA mix was covered with a hydrophobic 13-mm diameter × 0.1 mm glass coverslips that were prepared fresh by coating them with PlusONE Repel-Silene ES (GE Healthcare) for 15 min at room temperature and dried with an air pistol. Polymerization proceeded for 45 min at room temperature in a humidifier chamber. The coverslip was carefully removed, and gels were washed three times for 2 min with 10 mM HEPES.

Acrylamide, bisAcrylamide, Dithiothreitol (DTT), iodoacetamide, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), Sodium Dodecyl Sulfate (SDS), urea, glycerol, thiourea, Tetramethylethylenediamine (TEMED), Ammonium Persulfate (APS), molecular markers and Immobilized pH Gradient (IPG) buffer were obtained from GE Healthcare Life Sciences (São Paulo, SP, Brazil). Triton X-100, Bovine Serum Albumin (BSA) and Coomassie Brilliant Blue (CBB) were obtained from Sigma-Aldrich (São Paulo, SP, Brazil). Trypsin was obtained from Promega (São Paulo, SP, Brazil).