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4, were pooled, dialyzed against buffer B to a conductivity equivalent to that of buffer B containing 0.07 M KCl, and applied to a TSK DEAE-NPR (35 mm by 4 6 mm; Toso-Haas, Montgomeryville, PA) equilibrated in the same buffer. The column was eluted at 0 3 ml/min with a linear gradient from 0.07 to 0.5 M KCl in buffer B Fractions (0.1 ml) were collected, and the active fractions, which eluted with ∼0.2 M KCl, were pooled, diluted with an equal volume of 4 M guanidine hydrochlonde, 4 M urea, 7.5% acetonitrile, O 15% trifluoroacetic acid (TFA), and O 2% Zwittergent ZC-8 (Calbiochem, La Jolla, CA), and applied at 0 1 ml/min to a PLRP-S(1 mm by 50 mm; pore size 1000 Å, particle size 8 μm) rpHPLC column (Michrom BioResources, Auburn, CA) preequilibrated at 40°C in 2% eluant B (90% acetonitrile, 0.09% TFA) and 98% eluant A (2% acetonitrile, 0.1% TFA). The column was developed at 0 1 ml/min with a 2-min linear gradient from 2 to 25% eluant B, followed by a 23-min linear gradient from 25 to 60% eluant B.
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Approximately 50 pmol of PLRP-S-punfied rat p80 (6) was reduced, S-carboxyamidomethylated. digested with trypsin, and fractionated by microbore HPLC. Optimal peptides were determined by differential absorbance of ultraviolet light and matrix assisted laser de sorption mass spectrometry (Lasermat; Finnigan-MAT, San Jose, CA). The peptides were then sequenced by automated Edman degradation [W S Lane, A. Galat, M. W. Harding, S. L Schreiber, J. Protein Chem 10, 151 (1991)].
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13344257394
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note
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4. 1 mM DTT, and 10% (v/v) glycerol After 90 min on ice, the protein was dialyzed for 3 hours at 4°C against the same buffer without DTT
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D R Duan et al , Science 269, 1402 (1995); T. Aso, W. S. Lane, J W. Conaway, R. C Conaway, ibid., p 1439, A Kibel, O. lliopoulos, J. A. DeCaprio, W. G Kaelin Jr, ibid., p. 1444.
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D R Duan et al , Science 269, 1402 (1995); T. Aso, W. S. Lane, J W. Conaway, R. C Conaway, ibid., p 1439, A Kibel, O. lliopoulos, J. A. DeCaprio, W. G Kaelin Jr, ibid., p. 1444.
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27
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13344274078
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note
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We thank R. Robinson of the Harvard Microchemistry Facility for peptide isolation. Supported by NIH grant GM41628 and by funds provided to the Oklahoma Medical Research Foundation by the H. A and Mary K. Chapman Charitable Trust (A.S , R.C.C , and J.W.C) and by the William K. Warren Foundation (K W J) AS is supported by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research.
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