Mapping of radiolabeled peptides derived from proteolysis of polypeptides bound to nitrocellulose after “Western” blotting
Sections of nitrocellulose containing bound 32P-labeled polypeptides were excised from “Western” blots and exhaustively digested by trypsin in order to analyze the distribution of phosphorylation sites between the products of limited proteolysis of the multifunctional protein CAD. Using the criterio...
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| Veröffentlicht in: | Anal. Biochem.; (United States) 1986-11, Vol.158 (2), p.431-435 |
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| creator | Carrey, Elizabeth A. Hardie, D.Grahame |
| description | Sections of nitrocellulose containing bound
32P-labeled polypeptides were excised from “Western” blots and exhaustively digested by trypsin in order to analyze the distribution of phosphorylation sites between the products of limited proteolysis of the multifunctional protein CAD. Using the criterion of analytical isoelectric focusing, the
32P-peptides obtained by this method were found to be similar, although not identical, to peptides obtained by a more conventional digestion of trichloroacetic acid precipitates. Digestion on Western blots is more straightforward than electrophoretic elution of individual gel slices, gives better recoveries than direct digestion of gel slices, and is particularly suitable for peptide mapping of small peptides which bind to nitrocellulose but would diffuse out of polyacrylamide gels during the commonly used fixing and staining procedures. |
| doi_str_mv | 10.1016/0003-2697(86)90571-3 |
| format | Article |
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32P-labeled polypeptides were excised from “Western” blots and exhaustively digested by trypsin in order to analyze the distribution of phosphorylation sites between the products of limited proteolysis of the multifunctional protein CAD. Using the criterion of analytical isoelectric focusing, the
32P-peptides obtained by this method were found to be similar, although not identical, to peptides obtained by a more conventional digestion of trichloroacetic acid precipitates. Digestion on Western blots is more straightforward than electrophoretic elution of individual gel slices, gives better recoveries than direct digestion of gel slices, and is particularly suitable for peptide mapping of small peptides which bind to nitrocellulose but would diffuse out of polyacrylamide gels during the commonly used fixing and staining procedures.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1016/0003-2697(86)90571-3</identifier><identifier>PMID: 3544949</identifier><identifier>CODEN: ANBCA2</identifier><language>eng</language><publisher>San Diego, CA: Elsevier Inc</publisher><subject>550201 - Biochemistry- Tracer Techniques ; Analytical, structural and metabolic biochemistry ; Applied sciences ; Aspartate Carbamoyltransferase ; ATP ; AUTORADIOGRAPHY ; BASIC BIOLOGICAL SCIENCES ; BETA DECAY RADIOISOTOPES ; BETA-MINUS DECAY RADIOISOTOPES ; Biological and medical sciences ; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) ; CARBOHYDRATES ; CELLULOSE ESTERS ; CHEMICAL EXPLOSIVES ; CHEMICAL REACTIONS ; Collodion ; DAYS LIVING RADIOISOTOPES ; DECOMPOSITION ; Dihydroorotase ; ELECTROPHORESIS ; Electrophoresis, Polyacrylamide Gel - methods ; electrophoretic blotting ; ENZYMATIC HYDROLYSIS ; ENZYME ACTIVITY ; ENZYMES ; ESTERS ; Exact sciences and technology ; EXPLOSIVES ; Fundamental and applied biological sciences. Psychology ; gel electrophoresis, proteins ; General aspects, investigation methods ; HYDROLYSIS ; isoelectric focusing ; Isoelectric Point ; ISOTOPES ; LABELLED COMPOUNDS ; LIGASES ; LIGHT NUCLEI ; LYSIS ; Multienzyme Complexes ; Neoplasm Proteins - analysis ; NITRIC ACID ESTERS ; NITROCELLULOSE ; NUCLEI ; NUCLEOTIDES ; ODD-ODD NUCLEI ; ORGANIC COMPOUNDS ; Other techniques and industries ; Peptide Fragments - analysis ; Peptide Hydrolases - metabolism ; PEPTIDES ; Phosphoproteins - analysis ; PHOSPHORUS 32 ; PHOSPHORUS ISOTOPES ; PHOSPHORYLATION ; POLYPEPTIDES ; POLYSACCHARIDES ; proteases ; protein kinases ; PROTEINS ; RADIOISOTOPES ; SACCHARIDES ; SOLVOLYSIS</subject><ispartof>Anal. Biochem.; (United States), 1986-11, Vol.158 (2), p.431-435</ispartof><rights>1986</rights><rights>1987 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c442t-9426d177e92a288ccf8cae265847b0a7993e7e10e16dcdaa928f5d34948f9c63</citedby><cites>FETCH-LOGICAL-c442t-9426d177e92a288ccf8cae265847b0a7993e7e10e16dcdaa928f5d34948f9c63</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/0003269786905713$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,881,3537,27903,27904,65309</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=8378480$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=8381777$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/3544949$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://www.osti.gov/biblio/5878204$$D View this record in Osti.gov$$Hfree_for_read</backlink></links><search><creatorcontrib>Carrey, Elizabeth A.</creatorcontrib><creatorcontrib>Hardie, D.Grahame</creatorcontrib><creatorcontrib>Univ. of Dundee, Scotland</creatorcontrib><title>Mapping of radiolabeled peptides derived from proteolysis of polypeptides bound to nitrocellulose after “Western” blotting</title><title>Anal. Biochem.; (United States)</title><addtitle>Anal Biochem</addtitle><description>Sections of nitrocellulose containing bound
32P-labeled polypeptides were excised from “Western” blots and exhaustively digested by trypsin in order to analyze the distribution of phosphorylation sites between the products of limited proteolysis of the multifunctional protein CAD. Using the criterion of analytical isoelectric focusing, the
32P-peptides obtained by this method were found to be similar, although not identical, to peptides obtained by a more conventional digestion of trichloroacetic acid precipitates. Digestion on Western blots is more straightforward than electrophoretic elution of individual gel slices, gives better recoveries than direct digestion of gel slices, and is particularly suitable for peptide mapping of small peptides which bind to nitrocellulose but would diffuse out of polyacrylamide gels during the commonly used fixing and staining procedures.</description><subject>550201 - Biochemistry- Tracer Techniques</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Applied sciences</subject><subject>Aspartate Carbamoyltransferase</subject><subject>ATP</subject><subject>AUTORADIOGRAPHY</subject><subject>BASIC BIOLOGICAL SCIENCES</subject><subject>BETA DECAY RADIOISOTOPES</subject><subject>BETA-MINUS DECAY RADIOISOTOPES</subject><subject>Biological and medical sciences</subject><subject>Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)</subject><subject>CARBOHYDRATES</subject><subject>CELLULOSE ESTERS</subject><subject>CHEMICAL EXPLOSIVES</subject><subject>CHEMICAL REACTIONS</subject><subject>Collodion</subject><subject>DAYS LIVING RADIOISOTOPES</subject><subject>DECOMPOSITION</subject><subject>Dihydroorotase</subject><subject>ELECTROPHORESIS</subject><subject>Electrophoresis, Polyacrylamide Gel - methods</subject><subject>electrophoretic blotting</subject><subject>ENZYMATIC HYDROLYSIS</subject><subject>ENZYME ACTIVITY</subject><subject>ENZYMES</subject><subject>ESTERS</subject><subject>Exact sciences and technology</subject><subject>EXPLOSIVES</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>gel electrophoresis, proteins</subject><subject>General aspects, investigation methods</subject><subject>HYDROLYSIS</subject><subject>isoelectric focusing</subject><subject>Isoelectric Point</subject><subject>ISOTOPES</subject><subject>LABELLED COMPOUNDS</subject><subject>LIGASES</subject><subject>LIGHT NUCLEI</subject><subject>LYSIS</subject><subject>Multienzyme Complexes</subject><subject>Neoplasm Proteins - analysis</subject><subject>NITRIC ACID ESTERS</subject><subject>NITROCELLULOSE</subject><subject>NUCLEI</subject><subject>NUCLEOTIDES</subject><subject>ODD-ODD NUCLEI</subject><subject>ORGANIC COMPOUNDS</subject><subject>Other techniques and industries</subject><subject>Peptide Fragments - analysis</subject><subject>Peptide Hydrolases - metabolism</subject><subject>PEPTIDES</subject><subject>Phosphoproteins - analysis</subject><subject>PHOSPHORUS 32</subject><subject>PHOSPHORUS ISOTOPES</subject><subject>PHOSPHORYLATION</subject><subject>POLYPEPTIDES</subject><subject>POLYSACCHARIDES</subject><subject>proteases</subject><subject>protein kinases</subject><subject>PROTEINS</subject><subject>RADIOISOTOPES</subject><subject>SACCHARIDES</subject><subject>SOLVOLYSIS</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1986</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc1qFTEUx4NY6rX6BgpBRHQxmsxk8rEpSPGjUHFTcBkyyYlG5k7GJFPoRvog9uX6JGa8l7u0qxxyfufj_z8IPaPkLSWUvyOEdE3LlXgt-RtFekGb7gHaUKJ4QzqiHqLNAXmEHuf8kxBKWc-P0XHXM6aY2qDfX8w8h-k7jh4n40IczQAjODzDXIKDjB2kcFU_fIpbPKdYII7XOeS1Yq7hARziMjlcIp5CSdHCOC5jzICNL5Dw3c2fb5BrNN3d3OJhjKXUsU_QkTdjhqf79wRdfvxwefa5ufj66fzs_UVjGWtLo1jLHRUCVGtaKa310hpoeS-ZGIgRSnUggBKg3FlnjGql711XJUqvLO9O0Itd25hL0NmGAvaHjdMEtuheCtkSVqFXO6iK_LXUXfU25FWGmSAuWQvR8mp8V0G2A22KOSfwek5ha9K1pkSvp9Gr73r1XUuu_51Gr2XP9_2XYQvuULS_Rc2_3OdNtmb0yUw25AMmO1kdEPdjQjJJKna6w6DaehUgrbJhsuBCWlW7GP6_7l-gprsM</recordid><startdate>19861101</startdate><enddate>19861101</enddate><creator>Carrey, Elizabeth A.</creator><creator>Hardie, D.Grahame</creator><general>Elsevier Inc</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>OTOTI</scope></search><sort><creationdate>19861101</creationdate><title>Mapping of radiolabeled peptides derived from proteolysis of polypeptides bound to nitrocellulose after “Western” blotting</title><author>Carrey, Elizabeth A. ; Hardie, D.Grahame</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c442t-9426d177e92a288ccf8cae265847b0a7993e7e10e16dcdaa928f5d34948f9c63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1986</creationdate><topic>550201 - Biochemistry- Tracer Techniques</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Applied sciences</topic><topic>Aspartate Carbamoyltransferase</topic><topic>ATP</topic><topic>AUTORADIOGRAPHY</topic><topic>BASIC BIOLOGICAL SCIENCES</topic><topic>BETA DECAY RADIOISOTOPES</topic><topic>BETA-MINUS DECAY RADIOISOTOPES</topic><topic>Biological and medical sciences</topic><topic>Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)</topic><topic>CARBOHYDRATES</topic><topic>CELLULOSE ESTERS</topic><topic>CHEMICAL EXPLOSIVES</topic><topic>CHEMICAL REACTIONS</topic><topic>Collodion</topic><topic>DAYS LIVING RADIOISOTOPES</topic><topic>DECOMPOSITION</topic><topic>Dihydroorotase</topic><topic>ELECTROPHORESIS</topic><topic>Electrophoresis, Polyacrylamide Gel - methods</topic><topic>electrophoretic blotting</topic><topic>ENZYMATIC HYDROLYSIS</topic><topic>ENZYME ACTIVITY</topic><topic>ENZYMES</topic><topic>ESTERS</topic><topic>Exact sciences and technology</topic><topic>EXPLOSIVES</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>gel electrophoresis, proteins</topic><topic>General aspects, investigation methods</topic><topic>HYDROLYSIS</topic><topic>isoelectric focusing</topic><topic>Isoelectric Point</topic><topic>ISOTOPES</topic><topic>LABELLED COMPOUNDS</topic><topic>LIGASES</topic><topic>LIGHT NUCLEI</topic><topic>LYSIS</topic><topic>Multienzyme Complexes</topic><topic>Neoplasm Proteins - analysis</topic><topic>NITRIC ACID ESTERS</topic><topic>NITROCELLULOSE</topic><topic>NUCLEI</topic><topic>NUCLEOTIDES</topic><topic>ODD-ODD NUCLEI</topic><topic>ORGANIC COMPOUNDS</topic><topic>Other techniques and industries</topic><topic>Peptide Fragments - analysis</topic><topic>Peptide Hydrolases - metabolism</topic><topic>PEPTIDES</topic><topic>Phosphoproteins - analysis</topic><topic>PHOSPHORUS 32</topic><topic>PHOSPHORUS ISOTOPES</topic><topic>PHOSPHORYLATION</topic><topic>POLYPEPTIDES</topic><topic>POLYSACCHARIDES</topic><topic>proteases</topic><topic>protein kinases</topic><topic>PROTEINS</topic><topic>RADIOISOTOPES</topic><topic>SACCHARIDES</topic><topic>SOLVOLYSIS</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Carrey, Elizabeth A.</creatorcontrib><creatorcontrib>Hardie, D.Grahame</creatorcontrib><creatorcontrib>Univ. of Dundee, Scotland</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>OSTI.GOV</collection><jtitle>Anal. Biochem.; (United States)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Carrey, Elizabeth A.</au><au>Hardie, D.Grahame</au><aucorp>Univ. of Dundee, Scotland</aucorp><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Mapping of radiolabeled peptides derived from proteolysis of polypeptides bound to nitrocellulose after “Western” blotting</atitle><jtitle>Anal. Biochem.; (United States)</jtitle><addtitle>Anal Biochem</addtitle><date>1986-11-01</date><risdate>1986</risdate><volume>158</volume><issue>2</issue><spage>431</spage><epage>435</epage><pages>431-435</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><coden>ANBCA2</coden><abstract>Sections of nitrocellulose containing bound
32P-labeled polypeptides were excised from “Western” blots and exhaustively digested by trypsin in order to analyze the distribution of phosphorylation sites between the products of limited proteolysis of the multifunctional protein CAD. Using the criterion of analytical isoelectric focusing, the
32P-peptides obtained by this method were found to be similar, although not identical, to peptides obtained by a more conventional digestion of trichloroacetic acid precipitates. Digestion on Western blots is more straightforward than electrophoretic elution of individual gel slices, gives better recoveries than direct digestion of gel slices, and is particularly suitable for peptide mapping of small peptides which bind to nitrocellulose but would diffuse out of polyacrylamide gels during the commonly used fixing and staining procedures.</abstract><cop>San Diego, CA</cop><pub>Elsevier Inc</pub><pmid>3544949</pmid><doi>10.1016/0003-2697(86)90571-3</doi><tpages>5</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0003-2697 |
| ispartof | Anal. Biochem.; (United States), 1986-11, Vol.158 (2), p.431-435 |
| issn | 0003-2697 1096-0309 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_77261013 |
| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | 550201 - Biochemistry- Tracer Techniques Analytical, structural and metabolic biochemistry Applied sciences Aspartate Carbamoyltransferase ATP AUTORADIOGRAPHY BASIC BIOLOGICAL SCIENCES BETA DECAY RADIOISOTOPES BETA-MINUS DECAY RADIOISOTOPES Biological and medical sciences Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) CARBOHYDRATES CELLULOSE ESTERS CHEMICAL EXPLOSIVES CHEMICAL REACTIONS Collodion DAYS LIVING RADIOISOTOPES DECOMPOSITION Dihydroorotase ELECTROPHORESIS Electrophoresis, Polyacrylamide Gel - methods electrophoretic blotting ENZYMATIC HYDROLYSIS ENZYME ACTIVITY ENZYMES ESTERS Exact sciences and technology EXPLOSIVES Fundamental and applied biological sciences. Psychology gel electrophoresis, proteins General aspects, investigation methods HYDROLYSIS isoelectric focusing Isoelectric Point ISOTOPES LABELLED COMPOUNDS LIGASES LIGHT NUCLEI LYSIS Multienzyme Complexes Neoplasm Proteins - analysis NITRIC ACID ESTERS NITROCELLULOSE NUCLEI NUCLEOTIDES ODD-ODD NUCLEI ORGANIC COMPOUNDS Other techniques and industries Peptide Fragments - analysis Peptide Hydrolases - metabolism PEPTIDES Phosphoproteins - analysis PHOSPHORUS 32 PHOSPHORUS ISOTOPES PHOSPHORYLATION POLYPEPTIDES POLYSACCHARIDES proteases protein kinases PROTEINS RADIOISOTOPES SACCHARIDES SOLVOLYSIS |
| title | Mapping of radiolabeled peptides derived from proteolysis of polypeptides bound to nitrocellulose after “Western” blotting |
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