Electrophoretic resolution and comparison of brain proteins

THE TECHNIQUE of polyacrylamide gel electrophoresis has been successfully applied to a variety of problems over the past several years. Recently, a method has been described (AMES, 1973) which employs a discontinuous SDS buffer system (LAEMMLI, 1970) in a thin slab polyacrylamide gel electrophoresis apparatus (REID & BIELESKI, 1968; STUDIER, 1973) for the resolution of bacterial membrane, periplasmic and soluble proteins. We report here the advantages of this method for resolution and comparative analysis of proteins from whole brain homogenates. C57B1/6J normal and quaking mutant mice (Jackson Laboratories, Bar Harbor, Maine), 11-weeks-old, were anaesthetized with ether and decapitated. The whole brain (including olfactory bulbs and medulla) was removed and rinsed in 0.9% NaCl solution and blotted dry. Each brain (approximately 0.4 g) was homogenized in 4 ml of sample buffer (0,064 M tris, pH 6.8; 5 % glycerol; 5 % j-mercaptoethanol; 2 % SDS) with a Potter Teflon homogenizer. A 1 ml aliquot of each homogenate was then diluted with 3 ml of sample buffer and heated in boiling water for 2 min (duringwhich time the solution becomes totally transparent). This solution was then centrifuged (Sorvall centrifuge; 5 min at lo00 g), the pellet discarded, and the supernatant retained for use. Protein determination (GEIGER & BESSMAN, 1972) revealed that 10 p1 of each supernatant contains approximately 30 pg of protein. Two very similar types of electrophoretic systems were used. The first system (Hoefer Scientific Instruments, San Francisco) sandwiches a 0.75 mm thick slab gel between two glass plates (30 cm long, 18 cm wide), which is then vertically mounted on a lucite support equipped with a continuous-flow cooling system and both upper and lower electrode buffer reservoirs. The technique, using an upper 5% stacking gel and a lower 9 % separating gel, is essentially that of STUDIER (1973), as modified by AMES (1973). In addition, we made a single modification. Voltage (300 V) was applied to the separating gel for 8 h (pre-run) before pouring the stacking gel. A lucite or Teflon ‘comb’ of the same thickness as the gel, with teeth 0.8 cm wide was inserted into the stacking gel after pouring. Following gel polymerization, removal of these teeth provided wells for loading the samples. Brain sample volumes were 20 p1 per well. A series of molecular weight standards were individually added (10 pg in 20 pl each) to two of the wells; the molecular weight assignments