Bromophenol blue

Acetonitrile, formic acid, tris(hydroxymethyl) aminomethane (Tris), HCl, trichloroacetic acid, urea, dithiothreitol (DTT), bromophenol blue sodium salt, SDS, acrylamide/bis-acrylamide 30% solution (37.5% T; 1% C), ammonium bicarbonate phosphate, glycerol, iodoacetamide, acetone, Coomassie Brilliant Blue G (CBB), silver blue, and trypsin singles (Proteomics grade kit) were purchased from Sigma (St. Louis, MO) and used as received. DeStreak Rehydration Solution, immobiline DryStrip Cover Fluid, and bromophenol blue were from GE Healthcare Biosciences (Uppsala, Sweden); rennet with a strength of 1/10,000 was bought at a local market.
Sixty samples of fresh PDO buffalo mozzarella, produced in Campania, were kindly furnished by Istituto Zooprofilattico (Portici, Napoli, Italy). Ten samples obtained from Campanian factories producing buffalo mozzarella under specific PDO requirements were used as control.

The IPG strips (13 cm pH 4-7) were passively rehydrated with the samples containing 600 g of protein for 14 h. Isoelectric focusing was performed in an IPGPhor III (GE Healthcare) system as follows: one step of 500 V for 1 h, one gradient to 1000 V for 1 h, one gradient to 8000 V for 4 h and one step of 8000 V for 6 h, accumulating a total of 67250 Vh. The IPG strips were than equilibrated in two steps, for 20 min each, using an equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M Urea, 30% (v/v) glycerol, 30% (w/v) SDS and trace amounts of bromophenol blue, all from GE Healthcare) containing 1% (w/v) DTT (GE Healthcare) or 2.5% (w/v) iodoacetamide (GE Healthcare). Equilibrated strips were submitted to the second dimension of electrophoresis on 12.5% (w/v) SDS-PAGE gels, according to Laemlli [19] , in an Ettan Dalt Six cube system (GE Healthcare), using Electrophoresis Power Supply -EPS601 at 10 mA per gel for 1 h, and then at 40 mA per gel until complete. Temperature was controlled by a MultiTemp III (GE Healthcare) at 15ЊC. An Amersham Low Molecular Weight Kit for SDS Eletrophoresis (GE Healthcare) was used as molecular weight marker. Gels were stained using PhastGel TM Blue R (GE Healthcare) according to the manu-facturer´s instructions.