Epitope mapping of antibodies against S-tagged fusion proteins and molecular weight markers
Monoclonal antibodies against S-tagged fusion proteins expressed in pET vectors were generated and further characterized. Most pET vectors contain a 15meric S-tag as a fusion tag for the detection of recombinant proteins. Two antibodies, G18BA3 and G18BE8, recognized this S-tag in enzyme immunoassay...
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| Veröffentlicht in: | Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2008-02, Vol.72 (2), p.346-351 |
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| creator | Daskalow, K.(University of Potsdam, Golm (Germany)) Boisguerin, P Jandrig, B Volkmer, R Micheel, B Schenk, J.A |
| description | Monoclonal antibodies against S-tagged fusion proteins expressed in pET vectors were generated and further characterized. Most pET vectors contain a 15meric S-tag as a fusion tag for the detection of recombinant proteins. Two antibodies, G18BA3 and G18BE8, recognized this S-tag in enzyme immunoassay and Western blot. Their epitopes were mapped using peptide array technology and were confirmed to be AAKFERQHMDSPD. This corresponds to the C-terminal region of the S-tag plus additional amino acids P and D, which are also present in most available pET vectors. Amino acid substitution analysis revealed several essential residues for binding. The binding motif was therefore FExxHxDxxD for G18BA3 and AxxFExxH for G18BE8. Since some commercially available protein standards are expressed in pET vectors, G18BA3 and G18BE8 were also found to detect the ladder bands of a molecular weight marker on immunoblot analysis. Both antibodies should be highly useful for the simultaneous detection of recombinant pET vector-expressed fusion proteins and protein molecular weight standards in Western blotting, especially when chemoluminescent detection systems are used. |
| doi_str_mv | 10.1271/bbb.70406 |
| format | Article |
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Most pET vectors contain a 15meric S-tag as a fusion tag for the detection of recombinant proteins. Two antibodies, G18BA3 and G18BE8, recognized this S-tag in enzyme immunoassay and Western blot. Their epitopes were mapped using peptide array technology and were confirmed to be AAKFERQHMDSPD. This corresponds to the C-terminal region of the S-tag plus additional amino acids P and D, which are also present in most available pET vectors. Amino acid substitution analysis revealed several essential residues for binding. The binding motif was therefore FExxHxDxxD for G18BA3 and AxxFExxH for G18BE8. Since some commercially available protein standards are expressed in pET vectors, G18BA3 and G18BE8 were also found to detect the ladder bands of a molecular weight marker on immunoblot analysis. Both antibodies should be highly useful for the simultaneous detection of recombinant pET vector-expressed fusion proteins and protein molecular weight standards in Western blotting, especially when chemoluminescent detection systems are used.</description><identifier>ISSN: 0916-8451</identifier><identifier>EISSN: 1347-6947</identifier><identifier>DOI: 10.1271/bbb.70406</identifier><identifier>PMID: 18256509</identifier><language>eng</language><publisher>Tokyo: Japan Society for Bioscience, Biotechnology, and Agrochemistry</publisher><subject>Amino Acid Sequence ; AMINO ACID SEQUENCES ; Animals ; Antibodies - chemistry ; Antibodies - immunology ; ANTICORPS MONOCLONAL ; ANTICUERPOS MONOCLONALES ; Biological and medical sciences ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; ENZIMAS ; ENZYME ; ENZYMES ; Epitope Mapping ; Fundamental and applied biological sciences. Psychology ; GENETIC TRANSFORMATION ; hybridoma ; IMMUNOENZYME TECHNIQUES ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; MOLECULAR WEIGHT ; molecular weight standard ; MONOCLONAL ANTIBODIES ; monoclonal antibody ; PEPTIDE ; PEPTIDES ; PEPTIDOS ; PESO MOLECULAR ; POIDS MOLECULAIRE ; PROTEINAS ; PROTEINAS RECOMBINANTES ; PROTEINE ; PROTEINE RECOMBINANTE ; PROTEINS ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - immunology ; RECOMBINANT PROTEINS ; SECUENCIAS DE AMINOACIDOS ; SEQUENCE D'ACIDES AMINES ; substitution analysis ; TECHNIQUE IMMUNOENZYMATIQUE ; TECNICAS INMUNOENZIMATICAS ; TRANSFORMACION GENETICA ; TRANSFORMATION GENETIQUE</subject><ispartof>Bioscience, biotechnology, and biochemistry, 2008-02, Vol.72 (2), p.346-351</ispartof><rights>2008 by Japan Society for Bioscience, Biotechnology, and Agrochemistry 2008</rights><rights>2008 INIST-CNRS</rights><rights>Copyright Japan Science and Technology Agency 2008</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c540t-fae9e3d878af329c2c171644a5e93a50b2e888ede65780d433b13ad3ffc98863</citedby><cites>FETCH-LOGICAL-c540t-fae9e3d878af329c2c171644a5e93a50b2e888ede65780d433b13ad3ffc98863</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=20232909$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18256509$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Daskalow, K.(University of Potsdam, Golm (Germany))</creatorcontrib><creatorcontrib>Boisguerin, P</creatorcontrib><creatorcontrib>Jandrig, B</creatorcontrib><creatorcontrib>Volkmer, R</creatorcontrib><creatorcontrib>Micheel, B</creatorcontrib><creatorcontrib>Schenk, J.A</creatorcontrib><title>Epitope mapping of antibodies against S-tagged fusion proteins and molecular weight markers</title><title>Bioscience, biotechnology, and biochemistry</title><addtitle>Biosci Biotechnol Biochem</addtitle><description>Monoclonal antibodies against S-tagged fusion proteins expressed in pET vectors were generated and further characterized. Most pET vectors contain a 15meric S-tag as a fusion tag for the detection of recombinant proteins. Two antibodies, G18BA3 and G18BE8, recognized this S-tag in enzyme immunoassay and Western blot. Their epitopes were mapped using peptide array technology and were confirmed to be AAKFERQHMDSPD. This corresponds to the C-terminal region of the S-tag plus additional amino acids P and D, which are also present in most available pET vectors. Amino acid substitution analysis revealed several essential residues for binding. The binding motif was therefore FExxHxDxxD for G18BA3 and AxxFExxH for G18BE8. Since some commercially available protein standards are expressed in pET vectors, G18BA3 and G18BE8 were also found to detect the ladder bands of a molecular weight marker on immunoblot analysis. Both antibodies should be highly useful for the simultaneous detection of recombinant pET vector-expressed fusion proteins and protein molecular weight standards in Western blotting, especially when chemoluminescent detection systems are used.</description><subject>Amino Acid Sequence</subject><subject>AMINO ACID SEQUENCES</subject><subject>Animals</subject><subject>Antibodies - chemistry</subject><subject>Antibodies - immunology</subject><subject>ANTICORPS MONOCLONAL</subject><subject>ANTICUERPOS MONOCLONALES</subject><subject>Biological and medical sciences</subject><subject>Blotting, Western</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>ENZIMAS</subject><subject>ENZYME</subject><subject>ENZYMES</subject><subject>Epitope Mapping</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>GENETIC TRANSFORMATION</subject><subject>hybridoma</subject><subject>IMMUNOENZYME TECHNIQUES</subject><subject>Mice</subject><subject>Mice, Inbred BALB C</subject><subject>Molecular Sequence Data</subject><subject>MOLECULAR WEIGHT</subject><subject>molecular weight standard</subject><subject>MONOCLONAL ANTIBODIES</subject><subject>monoclonal antibody</subject><subject>PEPTIDE</subject><subject>PEPTIDES</subject><subject>PEPTIDOS</subject><subject>PESO MOLECULAR</subject><subject>POIDS MOLECULAIRE</subject><subject>PROTEINAS</subject><subject>PROTEINAS RECOMBINANTES</subject><subject>PROTEINE</subject><subject>PROTEINE RECOMBINANTE</subject><subject>PROTEINS</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - immunology</subject><subject>RECOMBINANT PROTEINS</subject><subject>SECUENCIAS DE AMINOACIDOS</subject><subject>SEQUENCE D'ACIDES AMINES</subject><subject>substitution analysis</subject><subject>TECHNIQUE IMMUNOENZYMATIQUE</subject><subject>TECNICAS INMUNOENZIMATICAS</subject><subject>TRANSFORMACION GENETICA</subject><subject>TRANSFORMATION GENETIQUE</subject><issn>0916-8451</issn><issn>1347-6947</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0V1r1jAUB_AginucXvgBlIIoeNF58tImvZSxTWWg4O68CKftSc1sm5q0jH17455HBRG8CiS_c5KcP2NPOZxwofmbtm1PNCio77Edl0qXdaP0fbaDhtelURU_Yo9SugbIGxV_yI64EVVdQbNjX84Wv4aFigmXxc9DEVyB8-rb0HtKBQ7o57QWn8sVh4H6wm3Jh7lYYlgpn2TbF1MYqdtGjMUN-eHrmnvFbxTTY_bA4ZjoyWE9ZlfnZ1en78rLjxfvT99ell2lYC0dUkOyN9qgk6LpRMc1r5XCihqJFbSCjDHUU11pA72SsuUSe-lc1xhTy2P2at82P-r7Rmm1k08djSPOFLZkNUgJUKv_Qp6nJkwDGb74C16HLc75D5Yr1RjBdSWyer1XXQwpRXJ2iT5__dZysD9zsTkXe5dLts8PHbd2ov6PPASRwcsDwNTh6CLOnU-_nQCRR3Pn1N752YU44U2IY29XvB1D_FUk_3X_s32Zw2BxiFl9-CQADIDSNZc_AB2rr4E</recordid><startdate>20080201</startdate><enddate>20080201</enddate><creator>Daskalow, K.(University of Potsdam, Golm (Germany))</creator><creator>Boisguerin, P</creator><creator>Jandrig, B</creator><creator>Volkmer, R</creator><creator>Micheel, B</creator><creator>Schenk, J.A</creator><general>Japan Society for Bioscience, Biotechnology, and Agrochemistry</general><general>Japan Society for Bioscience Biotechnology and Agrochemistry</general><general>Oxford University Press</general><scope>FBQ</scope><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>7T5</scope><scope>H94</scope><scope>7X8</scope></search><sort><creationdate>20080201</creationdate><title>Epitope mapping of antibodies against S-tagged fusion proteins and molecular weight markers</title><author>Daskalow, K.(University of Potsdam, Golm (Germany)) ; Boisguerin, P ; Jandrig, B ; Volkmer, R ; Micheel, B ; Schenk, J.A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c540t-fae9e3d878af329c2c171644a5e93a50b2e888ede65780d433b13ad3ffc98863</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Amino Acid Sequence</topic><topic>AMINO ACID SEQUENCES</topic><topic>Animals</topic><topic>Antibodies - chemistry</topic><topic>Antibodies - immunology</topic><topic>ANTICORPS MONOCLONAL</topic><topic>ANTICUERPOS MONOCLONALES</topic><topic>Biological and medical sciences</topic><topic>Blotting, Western</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>ENZIMAS</topic><topic>ENZYME</topic><topic>ENZYMES</topic><topic>Epitope Mapping</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>GENETIC TRANSFORMATION</topic><topic>hybridoma</topic><topic>IMMUNOENZYME TECHNIQUES</topic><topic>Mice</topic><topic>Mice, Inbred BALB C</topic><topic>Molecular Sequence Data</topic><topic>MOLECULAR WEIGHT</topic><topic>molecular weight standard</topic><topic>MONOCLONAL ANTIBODIES</topic><topic>monoclonal antibody</topic><topic>PEPTIDE</topic><topic>PEPTIDES</topic><topic>PEPTIDOS</topic><topic>PESO MOLECULAR</topic><topic>POIDS MOLECULAIRE</topic><topic>PROTEINAS</topic><topic>PROTEINAS RECOMBINANTES</topic><topic>PROTEINE</topic><topic>PROTEINE RECOMBINANTE</topic><topic>PROTEINS</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - immunology</topic><topic>RECOMBINANT PROTEINS</topic><topic>SECUENCIAS DE AMINOACIDOS</topic><topic>SEQUENCE D'ACIDES AMINES</topic><topic>substitution analysis</topic><topic>TECHNIQUE IMMUNOENZYMATIQUE</topic><topic>TECNICAS INMUNOENZIMATICAS</topic><topic>TRANSFORMACION GENETICA</topic><topic>TRANSFORMATION GENETIQUE</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Daskalow, K.(University of Potsdam, Golm (Germany))</creatorcontrib><creatorcontrib>Boisguerin, P</creatorcontrib><creatorcontrib>Jandrig, B</creatorcontrib><creatorcontrib>Volkmer, R</creatorcontrib><creatorcontrib>Micheel, B</creatorcontrib><creatorcontrib>Schenk, J.A</creatorcontrib><collection>AGRIS</collection><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>Immunology Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Bioscience, biotechnology, and biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Daskalow, K.(University of Potsdam, Golm (Germany))</au><au>Boisguerin, P</au><au>Jandrig, B</au><au>Volkmer, R</au><au>Micheel, B</au><au>Schenk, J.A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Epitope mapping of antibodies against S-tagged fusion proteins and molecular weight markers</atitle><jtitle>Bioscience, biotechnology, and biochemistry</jtitle><addtitle>Biosci Biotechnol Biochem</addtitle><date>2008-02-01</date><risdate>2008</risdate><volume>72</volume><issue>2</issue><spage>346</spage><epage>351</epage><pages>346-351</pages><issn>0916-8451</issn><eissn>1347-6947</eissn><abstract>Monoclonal antibodies against S-tagged fusion proteins expressed in pET vectors were generated and further characterized. Most pET vectors contain a 15meric S-tag as a fusion tag for the detection of recombinant proteins. Two antibodies, G18BA3 and G18BE8, recognized this S-tag in enzyme immunoassay and Western blot. Their epitopes were mapped using peptide array technology and were confirmed to be AAKFERQHMDSPD. This corresponds to the C-terminal region of the S-tag plus additional amino acids P and D, which are also present in most available pET vectors. Amino acid substitution analysis revealed several essential residues for binding. The binding motif was therefore FExxHxDxxD for G18BA3 and AxxFExxH for G18BE8. Since some commercially available protein standards are expressed in pET vectors, G18BA3 and G18BE8 were also found to detect the ladder bands of a molecular weight marker on immunoblot analysis. Both antibodies should be highly useful for the simultaneous detection of recombinant pET vector-expressed fusion proteins and protein molecular weight standards in Western blotting, especially when chemoluminescent detection systems are used.</abstract><cop>Tokyo</cop><pub>Japan Society for Bioscience, Biotechnology, and Agrochemistry</pub><pmid>18256509</pmid><doi>10.1271/bbb.70406</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 0916-8451 |
| ispartof | Bioscience, biotechnology, and biochemistry, 2008-02, Vol.72 (2), p.346-351 |
| issn | 0916-8451 1347-6947 |
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| source | J-STAGE Free; MEDLINE; Oxford University Press Journals All Titles (1996-Current); Freely Accessible Japanese Titles; EZB-FREE-00999 freely available EZB journals; Free Full-Text Journals in Chemistry |
| subjects | Amino Acid Sequence AMINO ACID SEQUENCES Animals Antibodies - chemistry Antibodies - immunology ANTICORPS MONOCLONAL ANTICUERPOS MONOCLONALES Biological and medical sciences Blotting, Western Electrophoresis, Polyacrylamide Gel ENZIMAS ENZYME ENZYMES Epitope Mapping Fundamental and applied biological sciences. Psychology GENETIC TRANSFORMATION hybridoma IMMUNOENZYME TECHNIQUES Mice Mice, Inbred BALB C Molecular Sequence Data MOLECULAR WEIGHT molecular weight standard MONOCLONAL ANTIBODIES monoclonal antibody PEPTIDE PEPTIDES PEPTIDOS PESO MOLECULAR POIDS MOLECULAIRE PROTEINAS PROTEINAS RECOMBINANTES PROTEINE PROTEINE RECOMBINANTE PROTEINS Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - immunology RECOMBINANT PROTEINS SECUENCIAS DE AMINOACIDOS SEQUENCE D'ACIDES AMINES substitution analysis TECHNIQUE IMMUNOENZYMATIQUE TECNICAS INMUNOENZIMATICAS TRANSFORMACION GENETICA TRANSFORMATION GENETIQUE |
| title | Epitope mapping of antibodies against S-tagged fusion proteins and molecular weight markers |
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