. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. OSMOTIC STRESS AND GENE EXPRESSION 333 Northern analysis Total RNA was isolated from the abdominal muscle and hepatopancreas samples (RNAgents kit, Promega), quanti- fied by absorbance at 260 nm with a spectrophotometer, and equally loaded (15 /u,g abdominal muscle total RNA; 25 p,g hepatopancreas total RNA) onto denaturing (formaldehyde) 1% agarose gels. These gels were washed (15 min, diethyl- pyrocarbonate water); blotted overnight onto nylon mem- branes (Magnagraph, MSI), UV cross-linked: and prehy- bridized (2 h) in 5X
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. OSMOTIC STRESS AND GENE EXPRESSION 333 Northern analysis Total RNA was isolated from the abdominal muscle and hepatopancreas samples (RNAgents kit, Promega), quanti- fied by absorbance at 260 nm with a spectrophotometer, and equally loaded (15 /u,g abdominal muscle total RNA; 25 p,g hepatopancreas total RNA) onto denaturing (formaldehyde) 1% agarose gels. These gels were washed (15 min, diethyl- pyrocarbonate water); blotted overnight onto nylon mem- branes (Magnagraph, MSI), UV cross-linked: and prehy- bridized (2 h) in 5X SSPE buffer ( M NaCl. 50 mM NaHiPO4, 5 mM ethylenediarninetetraacetic acid. pH ), 50% (w/v) formamide, 5X Denhardt's, 1% sodium dodecyl sulfate (SDS), and 100 jug/ml denatured sheared salmon sperm DNA at 42 °C. A partial lobster HSP90 clone (350 bp, cloning described in Chang etal., 1999) was 32P-labeled (Prime-It RmT. Stratagene), added directly to the prehybrid- ization solution, and allowed to hybridize overnight at 42 °C. Following hybridization, the blots were washed twice with 2X SSPE at room temperature and placed on film overnight at -70 °C. Following exposure of the film, the blots were stripped with several washes of buffer (15 mM NaCl. mM sodium citrate. pH 7. with SDS at 65 °C) until back- ground was minimal; they were then prehybridized. hybrid- ized, and washed as above, except that a partial lobster polyubiquitin cDNA probe was added (690 bp fragment from Ace I digest of lobster polyubiquitin clone; described in Shean and Mykles. 1995). To check for equal loading of RNA, the blots were probed with a partial lobster actin cDNA (700 bp. Harrison and El Haj. 1994). Lastly, follow- ing similar washes and prehybridization. a partial lobster HSP70 cDNA probe was hybridized with the blots (500 bp. Specs et al., 2002). Films were scanned on a high-resolution scanner, and densitometry was performed with NIH Image software. The signals from these northe
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