**3.5 Basic proteins constituents of the** *D. magna* **proteome**

Initially, single Daphnid extracts were analyzed on IPG strips with a pH range of 3-10. Since more than 80% of the proteins visualized by silver staining were in the pH 4-7 range, subsequent analyses were performed using IPGs pH 4-7. However, the theoretical 2D does predict a significant number of proteins (47%) in the basic range (7-10). Due to their relative low abundance, the visualization of very basic proteins (pH 7-10) in Daphnids required many more organisms. For this, 184 ± 3 mg of *D. magna* (approximately 135 organisms) were cultured under either normal or hypoxic conditions and processed in 1.3 mL of ProteoSOLVE IEF Reagent. The samples were concentrated two-fold by ultrafiltration, and IEF was performed on IPGs pH 7-10, followed by silver staining and image analysis. Silver staining detected 355 and 408 spots (gels not shown) in pH range 7-10 from *D. magna* cultured under normal or hypoxic conditions respectively, further illustrating the utility of 2-DE for detecting phenotypic differences influenced by specific environments.
