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For RNA isolation, rickettsial pellets were solubilized using 1 ml of Tri-Reagent (Applied Biosystems, Austin, TX) per-flask equivalent for L929-grown rickettsiae or per 0.5 ml of hen egg yolk sac rickettsial preparations.
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Six weeks later, hens at 31.7 weeks of age received a second immunization containing 0.60 mg total protein in both bacterins in 0.2 mL for hens.
The reaction mixture (5 mL) consisted of 0.2 mL of egg albumin (from fresh hen's egg), 2.8 mL of phosphate buffered saline (PBS, pH 6.4) and 2 mL of varying concentrations of extract so that final concentrations become 100, 200, 300, 400, 500 μg/mL.
The protein pellet was then dissolved in HENS buffer (1% SDS in HEN buffer; 0.1 mL of HENS buffer per mg of protein in the initial protein sample).
Item 26 My food preparation is simpler because of HEN.
Item 42 I have gained weight because of HEN.
Item 19 I have a good appetite because of HEN.
Lysozyme sensitivity assays were performed on LB medium plates containing different concentrations (0 to 20 mg/ml) of hen egg white lysozyme (HEWL) (Fluka, Buchs, Switzerland).
Cells having expressed His-tagged rRKIP were lysed in 50 mM Tris-HCl pH 8.0 by a combination of hen egg lysozyme (0.5 mg/ml) (Sigma) and sonication.
A 1-mg/mL stock solution of hen egg lysozyme (HEL; Sigma) was prepared in 0.1 mol/L phosphate buffer (pH 5.9) and aliquots were frozen until used.
A 1 mg/mL stock solution of hen egg lysozyme (HEL; Sigma, St . Louis MO) was prepared in 0.1 M phosphate buffer (pH 5.9), and aliquots were frozen until use.
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