Fractions collected from the Model 491 Prep Cell were analyzed by SDS-PAGE using the Mini-Protean II system. Starting with fraction 4, 60 l of every other fraction was mixed with 40 l of sample buffer (125 mM Tris-HCl (6.8), 30% glycerol, 4% b-mercaptoethanol, 4% SDS, and 0.001% bromophenol blue). After being electrophoresed on 7.5% polyacrylamide gels, separated proteins were electrophoretically blotted to PVDF membranes (5.5 x 8.5 cm) at a constant current of 2.0 mA/ cm2 of gel surface for 30 minutes. Blots were either stained with 0.05% Coomassie Blue or probed with antibody developed to tobacco BiP fusion protein (TOBBLP4, gift from Dr. J. Denecke).3
Preparative Isoelectric Focusing
Fractions 35, 36, and 37 from the Model 491 Prep Cell run were pooled and precipitated with 80% AS. The resulting 80% AS pellet was resuspended in 10 ml of dd H2O and dialyzed overnight against the same. The dialyzed sample was then mixed with 40 ml of Rotofor buffer (1% Bio-Lyte 5/7 ampholyte, 1% urea, and 5% glycerol). The sample was focused on the Rotofor cell at a constant power of 10 W for 4 hours at 4 C. Initial voltage was 1,250 and reached the 2,000 volts limit after 1 hour run time. Twenty fractions were collected from the Rotofor cell and each was lyophilized to a final volume of 500 l. Lyophilized samples were analyzed by SDS-PAGE as previously described.
The data in Figu