strip was equilibrated with 50 mM Tris-HC1 pH 6.8, 6 M Urea, 30% (v/v) glycerol, 2% (w/v) SDS, 2.5 % iodoacetamide, and a trace amount of bromophenol blue. The strip was then sealed on top of a 10 x 10 cm, 4-20% Tris-glycine gel (Invitrogen; Carlsbad, CA) with warm 0.5% agarose made in 25 mM Tris pH 8.3, 192 mM Glycine, and 0.1% SDS. The gel was run for 20 minutes at 20 V to load the sample and then an additional 90 min at 125 V to resolve the organelle proteins. After electrophoresis, the organelle proteins were transferred to an Immobilon P membrane (Millipore; Billerica, MA) by trans-blotting (14.5 hrs at 20 V and 4C) and stained with Coomassie brilliant blue R-250. N-terminal sequencing of the organelle proteins separated by 2D-PAGE was performed by the University of Florida, Interdisciplinary Center for Biotechnology Research, Protein Chemistry Core Facility using an Applied Biosystems model 494 HT sequencer and standard blot cartridge cycles.

Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS)

     The protein components of purified polyhedral organelles (115 [tg) were separated by 2D-IEF-SDS-PAGE as described above, but with the following modifications. The IEF dimension employed a 17 cm linear pH 4 to 7 IPG strip (Bio-Rad) and was focused for a total of 95 kV hours. After the IEF dimension, the IPG strip was sealed onto a 18.3 x19.3 cm, 8-16% polyacrylamide gel (Bio-Rad) and run for 20 minutes at 10 mA to load the sample and then another 5 to 6 hrs at 24 mA to resolve the organelle proteins. Following 2D-electrophoresis, the gel was stained with colloidal blue (Genomic Solutions, Ann Arbor, MI) and organelle proteins were excised, washed, dried, digested with trypsin, purified using a Millipore ZipTip, and then analyzed by MALDI-TOF-MS.