**2.3 Pressure Cycling Technology (PCT)**

PCT has been shown to be an effective means for isolating proteins from a variety of microorganisms, as well as many difficult-to-lyse samples such as *Caenorhabditis elegans* (Geiser et al., 2002; Smejkal et al., 2006b; Smejkal et al., 2007). PCT, which subjects samples to rapid cycles of pressure, facilitated the extraction of proteins from single *Daphnia magna* with and without ephippia and from single *Daphnia pulex*.

*Daphnia* were transferred to tared PULSE Tubes (Pressure BioSciences, Inc., South Easton, MA, USA) and suspended in 500 uL of 7M urea, 2M thiourea, and 4% CHAPS supplemented with 100 mM dithiothreitol (DTT) and protease inhibitor cocktail P-2714 (Sigma Aldrich Chemicals, St. Louis, MO). An additional 900 uL of mineral oil was added to accommodate the necessary volume for the PULSE Tubes. The tubes were placed in the Barocycler NEP-3229 (Pressure BioSciences, Inc., South Easton, MA, USA) for 60 pressure

Two-Dimensional Gel Electrophoresis Reveals

in Xinb3 clone (right).

Differential Protein Expression Between Individual *Daphnia* 319

describe above. Silver staining detected an average of 687 + 11 protein spots from the Xinb3 gels and 692 + 14 protein spots from the Iinb1 gels (figure 1). After normalization of the gel images based on total intensity, 679 spots were matched between the two gel images. One Hundred thirty six spots showed a two-fold or greater difference in spot intensity. Seventynine of these were more abundant in Xinb3 and 57 were more abundant in the Iinb1. To illustrate these differences, the silver stained gels were digitally colored. The Iinb1 gels were

red and the Xinb3 gels were green. The gels were then superimposed. (Figure 2).

Fig. 1. Representative silver-stained 2D gels of individual *Daphnia magna* with distinct genotypes. Quadruplicate gels revealed 692 + 14 spots in Iinb1 clone (left) and 687 + 11 spots

Fig. 2. Digitally enhanced, superimposed, silver-stained 2D gels of individual *Daphnia magna* with distinct genotypes. Red indicates spots unique to Iinb1, green indicates spots unique to

Xinb3, yellow indicate spots shared by both genotypes.

cycles, each cycle consisting of 10 seconds at 35,000 psi followed by rapid depressurization and held for 2 seconds at atmospheric pressure. Following PCT, each PULSE Tube was coupled to an Ultrafree-CL centrifugal filtration device with a 5-micron pore size (Millipore Corporation, Danvers, MA, USA) and evacuated by centrifugation for 1 minute at 1000 RCF. The PULSE Tube was removed and centrifugation continued for 4 minutes at 4000 RCF, followed by the removal of the mineral oil.
