A new quinoline-based acylhydrazone (1) has been synthesized and applied as a fluorescent probe. Probe 1 exhibits high selectivity and sensitivity to Cu2+ with fluorescence “ON-OFF ” behavior in HEPES buffered (1‰ DMSO, HEPES 20 mM, pH = 7.4) solution. The on-site generated 1-Cu2+ complex displays

were harvested by centrifugation at 850 ϫ g at 4°C for 5 min, washed twice with phosphate-buffered saline, and lysed in 0.5 ml of ice-cold lysis buffer (40 mM HEPES, pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 1.5 mM Na 3 Vo 4 , 1% Triton X-100, and one

]. In particular, bated on ice with swell buVer (10 mM Hepes, pH 7.2, 15 mM KCl), scraped into break buVer (50 mM Hepes, pH 7.2, 110 mM KCl), and washed with break buVer by centri-fugation at 800g for 5 min at 4 °C. Cells were then brought to volume in GGA buVer (25 mM Hepes, pH 7.4, 38 mM K-

2776 Labeling Conditions To label the plasma membrane, the basolateral side of the cells was incubated on ice with 50 g/ml Tf-HRP in internalization medium (IM) (of G-MEM, 10 mM Hepes pH 7.4, 5 mM glucose) containing 2 mg/ml BSA (IM/BSA). Unbound label was removed by two washes with ice-cold PBSϩ

with Hanks balanced salt solution (HBSS, Invitrogen) containing 10 mM HEPES (pH 7.4) and 0.2 mM EGTA, and then were digested with 0.1 % collagenase (Sigma) in HBSS with 10 mM HEPES, 2.5 mM CaCl2, and 25 mg/ml trypsin inhibitor. The perfused liver was gently removed and filtered through 70µm cell strainer

34. The soluble mouse T22b heavy chain and human b2M heterodimer complex was produced in E. coli and puriÞed as described (6). The T22b preparation was then concentrated to 13 mg/ml and extensively dia-lyzed against 20 mM Hepes (pH 7.2) and 25 mM sodium chloride before crystallization.

-buffered bath solution (137 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 10 mM D-Glucose, 5 mM HEPES, 20 µM 6-cyano-7nitroquinoxaline-2,3-dione (CNQX), 50 µM d(-)-2-amino-5-phosphonovaleric acid (AP5)). Acute brain slices (300 μm) were prepared from 3 wk Sprague-Dawley rats in accordance with national animal

-interacting protein; PMA, phorbol 12-myristate 13-acetate; buffer A, 119 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 2.6 mM CaCl2, 1.2 mM MgSO4, and 32.3 mM Hepes, pH 7.4, 2 mM glucose, 20 mg/ml BSA, and 200 nM adenosine; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; BSA, bovine serum albumin; SDS, sodium

Surface-sterilized oocysts of Cryptosporidium parvum were applied to subconfluent monolayers of human adenocarcinoma (HCT-8) cells grown on coverslips in six-well cluster plates. Parasite-infected cultures were then incubated in RPMI 1640 with 10 % fetal bovine serum, 15 mM HEPES (N-2

as described (Kinoshita et al., 2001). Quantitative RT-PCR for pIgR Expression Small intestines were collected from naïve or infected mice and flushed with sterile PBS. PP were removed and intestines were opened longitudinally, washed once with DTT (1mM in PBS) and incubated with cold HEPES/HBSS solution (150mM