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Volumn 278, Issue 5335, 1997, Pages 110-113

Interdomain interactions underlying activation of cyclic nucleotide- gated channels

Author keywords

[No Author keywords available]

Indexed keywords

AMINO TERMINAL SEQUENCE; ARTICLE; CATTLE; CHANNEL GATING; MOLECULAR INTERACTION; NONHUMAN; PRIORITY JOURNAL; PROTEIN PROTEIN INTERACTION; PROTEIN TARGETING;

EID: 0030863675     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.278.5335.110     Document Type: Article
Times cited : (135)

References (38)
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    • 6 or ThioHis fusions) strains; protein expression was induced by 0.25 mM isopropyl-β-D-thiogalactopyranoside at 25 to 30°C for 3 to 5 hours. Cells were harvested and resuspended in buffer S [50 mM tris-HCl (pH 7.8), 150 mM NaCl, 25 mM imidazole, 1% NDSB-256 (Calbiochem), 0.5% CHAPS detergent, and 0.25% Tween-20] containing protease inhibitors. Bacteria were lysed with a French press. Soluble protein was isolated by centrifugation at 20,000g at 4°C for 20 min. Glutathione-Sepharose beads (Pharmacia) were used to purify GST fusion proteins; His-tagged or ThioHis fusion proteins were purified with Ni-NTA beads (Qiagen) and eluted with 100 mM EDTA in buffer S. The ability of different recombinant proteins to interact was assayed by mixing various pairwise combinations in 0.5 ml of buffer S at 4°C overnight with shaking. The final concentration of immobilized GST fusion protein was about 25 to 50 μM (5 to 10 μl of glutathione-Sepharose beads; Pharmacia); the concentration of input protein was about 0.5 to 1 μM. Similar amounts of input protein were used for each assay as indicated in the input only blot. Beads were centrifuged at 2000g for 2 min and washed five times with 0.5 ml of buffer S. After the final wash, beads were resuspended in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer containing 2-mercaptoethanol, and bound proteins were separated by using denaturing 10 to 20% PAGE (Bio-Rad). After transfer to nitrocellulose, interacting input protein was visualized by using M2 monoclonal antibody (Eastman Kodak) directed against the FLAG epitope and the ECL chemiluminescence detection system (Amersham). Interaction assays were repeated at least three times with reproducible results.
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    • note
    • 2+ concentration) (2). Intracellular solutions containing cyclic nucleotides were changed with an RSC-100 rapid solution changer (Molecular Kinetics). Currents in the absence of cyclic nucleotide were subtracted.
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    • note
    • We thank K. Black, H. Utsugi, and G. Sheridan for expert technical assistance. We also gratefully acknowledge J. Hurley, M. Shapiro, G. Eaholtz, A. Fodor, and L. Sunderman for comments on the manuscript, and C. Tancioco for stimulating input. Supported by a grant from the National Eye Institute (EY 10329 to W.N.Z.). W.N.Z. is an Investigator and M.D.V. is an Associate of the Howard Hughes Medical Institute.


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