**4. Conclusion**

324 Gel Electrophoresis – Advanced Techniques

Fig. 7. Distribution of highly expressed *D. pulex* genes across 25 KOG classes. Blue and

red bars indicate the number of genes in each class from the top 2000 and 1000, respectively, most highly expressed genes (Left-hand axis). Green bars indicate the total genes assigned to each KOG class as a percentage of the total number of genes (18,371) assigned to a KOG class (Right-hand axis). To further characterize the predicted protein gel, we compared it to our observed spot counts. Table 1 summarizes the number

of predicted proteins and the number of actual proteins within specific pI ranges. Through the generation of several *D. magna* (both single animal and multiple-animal) 2D gels (not shown), we were able to visualize a total of 1285 protein spots. Seventy percent of these were observed in the 4-7 pI range, while the theoretical gel predicted 51% in this same range. It is important to note that the theoretical 2D does not account for post-translational modifications and was generated from *D. pulex* genes. Both of these factors will contribute to differences between the predicted (*D. pulex*) and observed

(*D. magna*) number of proteins.

Organisms live in ever changing environments. Understanding how individuals respond and adapt to these environments on a molecular level forms the basis for advances in personalized medicine and requires model systems with both well-developed ecological knowledge and molecular resources. The freshwater crustacean *Daphnia* now has these two requirements in place. We have demonstrated the ability of 2-DE to identify protein differences between single *Daphnia magna* with distinct genotypes (Iinb1 and Xinb3), distinct phenotypic differences (presence or absence of ephippia) and when cultured in different environments (normal or hypoxic conditions).

We predict that the detectable proteins on a single animal 2D gel, while biased towards the most abundant proteins, represent a functionally diverse set of proteins. This technique represents an important step to a greater understanding of individual variation of gene expression and is critical to advancing the field of EEFG. However, as the use of silver stain convolutes downstream mass spectrometry, the number of protein spots that can be confidently identified is significantly curtailed. The full potential of single animal gels will be realized with the development of comprehensive 2D maps of the *Daphnia* proteome.
