caa a22 4500 397556993 CHVBK 20180308164805.0 cr unu---uuuuu 161202e199607 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021389 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021389 Systematic Peptide Fragmentation of Polyvinylidene Difluoride(PVDF)-Immobilized Proteins Prior to Microsequencing [Elektronische Daten] [Akihiro Iwamatsu, Nobuko Yoshida-Kubomura] The sequential in situ digestion of proteins immobilized on polyvinylidene difluoride (PVDF) has been systematically designed and optimized. The method consists of immobilization of the proteins on PVDF, S-carboxymethylation, and then successive in situ digestions using specific proteases. In order to obtain high yields of the peptide fragments, from which specific amino acid residues connected to the N- or C-terminal of the resulting digestion fragments can be deduced, the cleavages are performed in the following order: (1) Achromobacter protease I (API), (2) endoproteinase Asp-N, and (3) trypsin. Procedures for recovering the numerous fragments remaining on the PVDF membrane after the third digestion with trypsin are also discussed. Application of sequential in situ digestion for the acquisition of fragments suitable for sequencing from digests of large-molecular-weight proteins is also presented. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence deducible amino acid residues nationallicence PVDF membrane nationallicence recoveries of digests from PVDF nationallicence S-alkylation of protein nationallicence sequential in situ digestion nationallicence Iwamatsu Akihiro Central Laboratories for Key Technology, Kirun Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236 aut Yoshida-Kubomura Nobuko Central Laboratories for Key Technology, Kirun Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236 aut The Journal of Biochemistry Oxford University Press 120/1(1996-07), 29-34 0021-924X 120:1<29 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021389 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021389 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Iwamatsu Akihiro Central Laboratories for Key Technology, Kirun Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236 aut NATIONALLICENCE 700 1- Yoshida-Kubomura Nobuko Central Laboratories for Key Technology, Kirun Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/1(1996-07), 29-34 0021-924X 120:1<29 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford