Appendix A. Field collection of Hsp70 samples and Western blot methods.
Tissue samples for Hsp70 analysis were taken from six Katharina each month from June 1999 to August 2000. Sampled individuals were outside experimental plots at the same tidal height. On each sampling occasion, I haphazardly selected 3 individuals from underneath the Hedophyllum canopy and 3 from unshaded areas, to avoid confounding effects of location on the evaluation of population Hsp70 levels. Hsp70 levels did not differ between animals collected in these microhabitats (P > 0.4 for all sampling dates), so data were pooled for analysis.
Little is known about fine temporal scale changes in Hsp production in natural populations. Studies with field populations of the intertidal mussel Mytilus trossulus have shown that Hsp synthesis following heat stress during low tide occurs primarily after re-immersion and continues for up to 16 h (Hofmann and Somero 1996) . Studies with an intertidal snail showed a similar pattern of Hsp synthesis following re-immersion (Tomanek and Somero 2000). Given these data, it is unlikely that the detected levels of Hsp70 in Katharina were affected by microhabitat selection on the same day on which samples were taken. As I had no way of knowing the location of freely ranging Katharina on previous low tides, these data are considered to be representative of the general physiological state of the Katharina population at Pile Point during the study period.
Western blots were run according to the Hofmann and Somero (1995) protocol with the following exceptions. For protein analysis, tissue samples were homogenized in 1 volume (w/v) of a 2% SDS homogenization buffer solution (50mM Tris-HCl (pH 6.8), 2% SDS, 1mM EDTA, 1mM PMSF). Homogenate was boiled for 7 minutes at 100°C and centrifuged for 10 minutes at 10,000g, and the supernatant was frozen at –80°C. Protein concentration of homogenates was determined by a modified Bradford protein assay (Pierce Coomassie Plus). For SDS- polyacrylamide electrophoresis, 15 mg of protein were mixed 1:1 (v/v) with Laemmli sample buffer (BioRad Laboratories) with 5% 2-mercaptoethanol, boiled for 3 min and centrifuged at 10,000g for 1 min. Samples were electophoresed on 10% SDS- polyacrylamide gels. On each gel I included a 0.05mg sample of purified bovine Hsc73 (Stressgen) as a positive control and standard to allow comparison of multiple western blots. Proteins were transferred to a nitrocellulose membrane via wet electrophoretic transfer at 30V for 15 hours, (transfer buffer = 192mM glycine, 25mM Tris, 20% methanol). Immunodetection was performed using an anti-Hsp70 rat monoclonal antibody that cross-reacts with multiple forms of Hsp70 (Affinity Bioreagents; MA3-001). Each blot was incubated for 1 min in 0.5 mL of ECL detection reagents (Amersham). Blots were exposed to x-ray film (Kodak X-OMAT AR 5) for 5 to 120 seconds. The developed film was scanned with a Fluor-S MultiImager System (BioRad Laboratories). Band intensities were quantified using Quantity One software (BioRad Laboratories).
LITERATURE CITED
Hofmann, G. E., and G. N. Somero. 1995. Evidence for protein damage at environmental temperatures: seasonal changes in levels of ubiquitin conjugates and hsp70 in the intertidal mussel Mytilus trossulus. Journal of Experimental Biology 198:1509–1518.
Hofmann, G. E., and G. N. Somero. 1996. Protein ubiquitination and stress protein synthesis in Mytilus trossulus occurs during recovery from tidal emersion. Molecular Marine Biology and Biotechnology 5:175–184.
Tomanek, L., and G. N. Somero. 2000. Time course and magnitude of synthesis of heat shock proteins in congeneric marine snails (Genus Tegula) from different tidal heights. Physiological and Biochemical Zoology 73:249–256.