maltoside was included in the isoelectric focusing step and the 2-D electrophoresis was repeated. Under these conditions single spots were observed for PduC, PduB, and PduB', but PduK, PduJ and PduP were still represented by multiple spots (data not shown). Hence, in the case of PduK, PduJ and PduP, post-translational modification is a possibility.

Densitometry, Molecular Mass, and pI Values of Polyhedral Organelle Proteins Separated by Two-Dimensional Electrophoresis

     Following 2D-electrophoresis of the purified organelles, the molecular mass, and pI values for each organelle protein were determined. In addition, protein spots were quantitated by densitometry so that the relative abundance of each organelle protein could be determined (Table 4-5).

     The pI values of organelle proteins were determined from the gel pictured in Figure 4-5, which utilized a linear gradient (pH 4-7). These values were then compared to pI values calculated from sequence analyses based on the protein identifications described above. In general, the observed and calculated pI values were similar. However, the pI values of PduA and PduD could not be accurately determined experimentally because both proteins focused at the upper limit of the pH range for the gel employed (pH 7) and both have calculated pI values above 7. Also spot 4 was not observed in the previous gels thus its identity and pI were not determined.

     Densitometry and molecular weight determination was performed on a 2D gel that included dodecyl-maltoside in the first-dimension (to reduce protein streaking) and that had been stained Sypro Ruby which has an extended linear range (Figure 4-7) (Berggren et al. 2000). Based on mass the PduA shell protein and the putative structural proteins PduBB'J by mass make up the bulk of the polyhedral organelle while PduKTU are minor