Discontinuous SDS-PAGE analysis was performed according to Laëmmli (1970). Separation of proteins by this procedure is mainly determined by their molecular weight. The anionic detergent SDS denatures the proteins by wrapping around the polypeptide backbone, and confers a net negative charge to the polypeptide that is proportional to its length. A high resolution of protein separation is obtained by the discontinuous system, because the polypeptides are first concentrated in a stacking gel before entering the separation gel.
Protein samples were prepared by adding 4X loading buffer (200 mM Tris.HCl pH 6.8, 8 mM EDTA, 40% glycerol, 4% SDS, 0.4% bromophenol blue, 6% thioglycerol), and boiling for 5 minutes. When total cell proteins had to be analysed, 0.5 ml of an E. coli culture in stationary growth phase was centrifuged to collect the cells, which were resuspended in 200 ml of loading buffer. Up to 20 ml of these solutions were loaded onto gels. LMW calibration mixtures (Pharmacia), consisting of phosphorylase b (94 kDa), albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), and a-lactalbumin (14.4 kDa) were used as marker for molecular mass.
The discontinuous gel was prepared by pouring a stacking gel (4% polyacrylamide in 125 mM Tris.HCl pH 6.8, 0.1% SDS) on top of a separation gel (12% to 15% polyacrylamide in 375 mM Tris.HCl pH 8.8, 0.1% SDS). Polymerization reactions were initiated by addition of 0.1% ammonium persulfate (the initiator peroxide) and N,N,N’,N’-tetramethylethylenediamine (TEMED, the catalyst) at 0.05% to the acrylamide/N,N’-methylenebisacrylamide (%C = 2.6) solutions (Bio-Rad Laboratories, Hercules, CA). Electrophoresis was performed in a MINI-PROTEAN II apparatus (Biorad, Hercules, CA) with tank buffer composed of 25 mM Tris.HCl pH 8.3, 192 mM glycine (UCB, Brussel, Belgium), 0.1% SDS. After separation, gels were stained with Coomassie brilliant blue R-250 (0.25%) in 45% methanol, 10% acetic acid, followed by destaining in 5% methanol, 7% acetic acid.
Protein bands were quantified with the BabyImager documentation system (Appligene, Illkirch, France) and Intelligent Quantifier™ software (B.I. Systems, Ann Arbor, MI).