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2642691356
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note
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4, 25 mg deoxyribonuclease 1. and 250 mM sucrose, plus pepstatin, leupeptin, and PMSF. The channel was extracted in the same solution containing 40 mM decylmaltoside (Anatrace) at room temperature. Following centrifugation the supernatant was bound to cobalt resin (Talon) at a protein to resin ratio that will saturate the resin. The resin was washed, and detergent concentration was lowered to 10.0 mM. One-milliliter columns were prepared. The control resin (no channel) was handled in the same manner. The resin preparation was the same for mass spectrometry and binding studies.
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17
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2642629381
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Forty milligrams of L. quinquestriatus hebraeus venom (Alomone Labs) was suspended in buffer identical to that of the channel (10.0 mM declymaltoside) and applied to the column. After washing, channel was eluted with 1.0 M imidazole in the same buffer
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Forty milligrams of L. quinquestriatus hebraeus venom (Alomone Labs) was suspended in buffer identical to that of the channel (10.0 mM declymaltoside) and applied to the column. After washing, channel was eluted with 1.0 M imidazole in the same buffer.
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85087323457
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J. A. Debin, J. E. Maggio, G. R. Strichartz, Am. J. Physiol. Soc. 264, C369 (1993); G. Lippens, J. Najib, S. J. Wodak, A. Tartar, Biochemistry 34, 13 (1995).
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23
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2642662151
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note
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D) of unlabeled wild-type agitoxin2 to determine nonspecific binding. The competition assay was carried out under the same conditions. Labeled agitoxin2 at 0.06 μM was always present and unlabeled toxin was added to compete with bound labeled toxin.
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26
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84889120137
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T. A. Jones, J. Y. Zou, J. Y. Cowan, M. Kjeldgaard, Acta Crystallogr. A47, 110 (1991).
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2642655994
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We thank J. Gulbis, J. P. Imredy, and D. A. Doyle for assistance in preparation of the figures and helpful discussions on the work. Supported by NIH GM43949. R.M. is an investigator in the Howard Hughes Medical Institute
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We thank J. Gulbis, J. P. Imredy, and D. A. Doyle for assistance in preparation of the figures and helpful discussions on the work. Supported by NIH GM43949. R.M. is an investigator in the Howard Hughes Medical Institute.
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