A standard operating procedure for an enzymatic activity inhibition assay
This Standard Operating Protocol (SOP) describes the key steps of experimental setup for an inhibition assay of enzymatic activity. The protocol begins with the design of an experiment, including the choice of a catalytic reaction, optimal conditions, fraction and concentration of the active enzyme,...
Gespeichert in:
| Veröffentlicht in: | European biophysics journal 2021-05, Vol.50 (3-4), p.345-352 |
|---|---|
| Hauptverfasser: | , , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | 352 |
|---|---|
| container_issue | 3-4 |
| container_start_page | 345 |
| container_title | European biophysics journal |
| container_volume | 50 |
| creator | Smirnovienė, Joana Baranauskienė, Lina Zubrienė, Asta Matulis, Daumantas |
| description | This Standard Operating Protocol (SOP) describes the key steps of experimental setup for an inhibition assay of enzymatic activity. The protocol begins with the design of an experiment, including the choice of a catalytic reaction, optimal conditions, fraction and concentration of the active enzyme, substrate and inhibitor concentrations and the positive and negative controls. The protocol ends with the data analysis followed by a typical example of an experiment. Despite an apparently standard procedure, the assay has a number of possible pitfalls such as low fraction of the active enzyme or errors in the analysis such as application of an improper model or incorrect determination of the inhibition constant while not recognizing the dependence on enzyme concentration. The protocol provides examples of necessary steps and controls to avoid these problems and obtain highly reliable results.
Graphic abstract |
| doi_str_mv | 10.1007/s00249-021-01530-8 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_webof</sourceid><recordid>TN_cdi_webofscience_primary_000640735700001</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>2539544570</sourcerecordid><originalsourceid>FETCH-LOGICAL-c375t-a4af77049c5fa57658306472017faebd7c10c4744e733c3020ec80ffbe910c8e3</originalsourceid><addsrcrecordid>eNqNkEtvFDEQhC0EIpvAH-CALHFBigbar_XMMVqRhxSJC5wtj6cnONr1LLaHaPPr6TAhSBwQPtiH_qpcXYy9EfBBANiPBUDqrgEpGhBGQdM-YyuhlWwECPucreg2jTVWHLHjUm4BtBGifcmOlGrXmjxW7OqMl-rT4PPApz1mX2O64fs8BRzmjHycMveJY7o_7GgWuA81_oj1wGP6FvtY45S4L8UfXrEXo98WfP34nrCv55--bC6b688XV5uz6yYoa2rjtR-tBd0FM3pj16ZVsNZWUtbRYz_YICBoqzVapYICCRhaGMceOxq0qE7Y-8WXQn6fsVS3iyXgdusTTnNx0gi9BtNJSei7v9Dbac6J0hGlOqO1sUCUXKiQp1Iyjm6f487ngxPgHop2S9GOina_inYtid4-Ws_9Docnye9mCThdgDvsp7GEiCngEwZAS4NV9D8dQXT7__QmVv_Q-2aaUyWpWqSF8HSD-c-S_8j_E1PFqC4</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>2539544570</pqid></control><display><type>article</type><title>A standard operating procedure for an enzymatic activity inhibition assay</title><source>SpringerNature Journals</source><source>Web of Science - Science Citation Index Expanded - 2021<img src="https://exlibris-pub.s3.amazonaws.com/fromwos-v2.jpg" /></source><creator>Smirnovienė, Joana ; Baranauskienė, Lina ; Zubrienė, Asta ; Matulis, Daumantas</creator><creatorcontrib>Smirnovienė, Joana ; Baranauskienė, Lina ; Zubrienė, Asta ; Matulis, Daumantas</creatorcontrib><description>This Standard Operating Protocol (SOP) describes the key steps of experimental setup for an inhibition assay of enzymatic activity. The protocol begins with the design of an experiment, including the choice of a catalytic reaction, optimal conditions, fraction and concentration of the active enzyme, substrate and inhibitor concentrations and the positive and negative controls. The protocol ends with the data analysis followed by a typical example of an experiment. Despite an apparently standard procedure, the assay has a number of possible pitfalls such as low fraction of the active enzyme or errors in the analysis such as application of an improper model or incorrect determination of the inhibition constant while not recognizing the dependence on enzyme concentration. The protocol provides examples of necessary steps and controls to avoid these problems and obtain highly reliable results.
Graphic abstract</description><identifier>ISSN: 0175-7571</identifier><identifier>EISSN: 1432-1017</identifier><identifier>DOI: 10.1007/s00249-021-01530-8</identifier><identifier>PMID: 33864100</identifier><language>eng</language><publisher>Cham: Springer International Publishing</publisher><subject>Assaying ; Biochemistry ; Biological and Medical Physics ; Biomedical and Life Sciences ; Biophysics ; Biophysics Letter ; Cell Biology ; Data analysis ; Enzymatic activity ; Enzymes ; Life Sciences ; Life Sciences & Biomedicine ; Membrane Biology ; Nanotechnology ; Neurobiology ; Science & Technology ; Substrate inhibition</subject><ispartof>European biophysics journal, 2021-05, Vol.50 (3-4), p.345-352</ispartof><rights>European Biophysical Societies' Association 2021</rights><rights>European Biophysical Societies' Association 2021.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>true</woscitedreferencessubscribed><woscitedreferencescount>5</woscitedreferencescount><woscitedreferencesoriginalsourcerecordid>wos000640735700001</woscitedreferencesoriginalsourcerecordid><citedby>FETCH-LOGICAL-c375t-a4af77049c5fa57658306472017faebd7c10c4744e733c3020ec80ffbe910c8e3</citedby><cites>FETCH-LOGICAL-c375t-a4af77049c5fa57658306472017faebd7c10c4744e733c3020ec80ffbe910c8e3</cites><orcidid>0000-0002-6178-6276 ; 0000-0002-9924-9177 ; 0000-0001-5996-1883 ; 0000-0001-5503-4775</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00249-021-01530-8$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00249-021-01530-8$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>315,781,785,27929,27930,39263,41493,42562,51324</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33864100$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Smirnovienė, Joana</creatorcontrib><creatorcontrib>Baranauskienė, Lina</creatorcontrib><creatorcontrib>Zubrienė, Asta</creatorcontrib><creatorcontrib>Matulis, Daumantas</creatorcontrib><title>A standard operating procedure for an enzymatic activity inhibition assay</title><title>European biophysics journal</title><addtitle>Eur Biophys J</addtitle><addtitle>EUR BIOPHYS J BIOPHY</addtitle><addtitle>Eur Biophys J</addtitle><description>This Standard Operating Protocol (SOP) describes the key steps of experimental setup for an inhibition assay of enzymatic activity. The protocol begins with the design of an experiment, including the choice of a catalytic reaction, optimal conditions, fraction and concentration of the active enzyme, substrate and inhibitor concentrations and the positive and negative controls. The protocol ends with the data analysis followed by a typical example of an experiment. Despite an apparently standard procedure, the assay has a number of possible pitfalls such as low fraction of the active enzyme or errors in the analysis such as application of an improper model or incorrect determination of the inhibition constant while not recognizing the dependence on enzyme concentration. The protocol provides examples of necessary steps and controls to avoid these problems and obtain highly reliable results.
Graphic abstract</description><subject>Assaying</subject><subject>Biochemistry</subject><subject>Biological and Medical Physics</subject><subject>Biomedical and Life Sciences</subject><subject>Biophysics</subject><subject>Biophysics Letter</subject><subject>Cell Biology</subject><subject>Data analysis</subject><subject>Enzymatic activity</subject><subject>Enzymes</subject><subject>Life Sciences</subject><subject>Life Sciences & Biomedicine</subject><subject>Membrane Biology</subject><subject>Nanotechnology</subject><subject>Neurobiology</subject><subject>Science & Technology</subject><subject>Substrate inhibition</subject><issn>0175-7571</issn><issn>1432-1017</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>HGBXW</sourceid><recordid>eNqNkEtvFDEQhC0EIpvAH-CALHFBigbar_XMMVqRhxSJC5wtj6cnONr1LLaHaPPr6TAhSBwQPtiH_qpcXYy9EfBBANiPBUDqrgEpGhBGQdM-YyuhlWwECPucreg2jTVWHLHjUm4BtBGifcmOlGrXmjxW7OqMl-rT4PPApz1mX2O64fs8BRzmjHycMveJY7o_7GgWuA81_oj1wGP6FvtY45S4L8UfXrEXo98WfP34nrCv55--bC6b688XV5uz6yYoa2rjtR-tBd0FM3pj16ZVsNZWUtbRYz_YICBoqzVapYICCRhaGMceOxq0qE7Y-8WXQn6fsVS3iyXgdusTTnNx0gi9BtNJSei7v9Dbac6J0hGlOqO1sUCUXKiQp1Iyjm6f487ngxPgHop2S9GOina_inYtid4-Ws_9Docnye9mCThdgDvsp7GEiCngEwZAS4NV9D8dQXT7__QmVv_Q-2aaUyWpWqSF8HSD-c-S_8j_E1PFqC4</recordid><startdate>20210501</startdate><enddate>20210501</enddate><creator>Smirnovienė, Joana</creator><creator>Baranauskienė, Lina</creator><creator>Zubrienė, Asta</creator><creator>Matulis, Daumantas</creator><general>Springer International Publishing</general><general>Springer Nature</general><general>Springer Nature B.V</general><scope>BLEPL</scope><scope>DTL</scope><scope>HGBXW</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0002-6178-6276</orcidid><orcidid>https://orcid.org/0000-0002-9924-9177</orcidid><orcidid>https://orcid.org/0000-0001-5996-1883</orcidid><orcidid>https://orcid.org/0000-0001-5503-4775</orcidid></search><sort><creationdate>20210501</creationdate><title>A standard operating procedure for an enzymatic activity inhibition assay</title><author>Smirnovienė, Joana ; Baranauskienė, Lina ; Zubrienė, Asta ; Matulis, Daumantas</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c375t-a4af77049c5fa57658306472017faebd7c10c4744e733c3020ec80ffbe910c8e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Assaying</topic><topic>Biochemistry</topic><topic>Biological and Medical Physics</topic><topic>Biomedical and Life Sciences</topic><topic>Biophysics</topic><topic>Biophysics Letter</topic><topic>Cell Biology</topic><topic>Data analysis</topic><topic>Enzymatic activity</topic><topic>Enzymes</topic><topic>Life Sciences</topic><topic>Life Sciences & Biomedicine</topic><topic>Membrane Biology</topic><topic>Nanotechnology</topic><topic>Neurobiology</topic><topic>Science & Technology</topic><topic>Substrate inhibition</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Smirnovienė, Joana</creatorcontrib><creatorcontrib>Baranauskienė, Lina</creatorcontrib><creatorcontrib>Zubrienė, Asta</creatorcontrib><creatorcontrib>Matulis, Daumantas</creatorcontrib><collection>Web of Science Core Collection</collection><collection>Science Citation Index Expanded</collection><collection>Web of Science - Science Citation Index Expanded - 2021</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>European biophysics journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Smirnovienė, Joana</au><au>Baranauskienė, Lina</au><au>Zubrienė, Asta</au><au>Matulis, Daumantas</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A standard operating procedure for an enzymatic activity inhibition assay</atitle><jtitle>European biophysics journal</jtitle><stitle>Eur Biophys J</stitle><stitle>EUR BIOPHYS J BIOPHY</stitle><addtitle>Eur Biophys J</addtitle><date>2021-05-01</date><risdate>2021</risdate><volume>50</volume><issue>3-4</issue><spage>345</spage><epage>352</epage><pages>345-352</pages><issn>0175-7571</issn><eissn>1432-1017</eissn><abstract>This Standard Operating Protocol (SOP) describes the key steps of experimental setup for an inhibition assay of enzymatic activity. The protocol begins with the design of an experiment, including the choice of a catalytic reaction, optimal conditions, fraction and concentration of the active enzyme, substrate and inhibitor concentrations and the positive and negative controls. The protocol ends with the data analysis followed by a typical example of an experiment. Despite an apparently standard procedure, the assay has a number of possible pitfalls such as low fraction of the active enzyme or errors in the analysis such as application of an improper model or incorrect determination of the inhibition constant while not recognizing the dependence on enzyme concentration. The protocol provides examples of necessary steps and controls to avoid these problems and obtain highly reliable results.
Graphic abstract</abstract><cop>Cham</cop><pub>Springer International Publishing</pub><pmid>33864100</pmid><doi>10.1007/s00249-021-01530-8</doi><tpages>8</tpages><orcidid>https://orcid.org/0000-0002-6178-6276</orcidid><orcidid>https://orcid.org/0000-0002-9924-9177</orcidid><orcidid>https://orcid.org/0000-0001-5996-1883</orcidid><orcidid>https://orcid.org/0000-0001-5503-4775</orcidid></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0175-7571 |
| ispartof | European biophysics journal, 2021-05, Vol.50 (3-4), p.345-352 |
| issn | 0175-7571 1432-1017 |
| language | eng |
| recordid | cdi_webofscience_primary_000640735700001 |
| source | SpringerNature Journals; Web of Science - Science Citation Index Expanded - 2021<img src="https://exlibris-pub.s3.amazonaws.com/fromwos-v2.jpg" /> |
| subjects | Assaying Biochemistry Biological and Medical Physics Biomedical and Life Sciences Biophysics Biophysics Letter Cell Biology Data analysis Enzymatic activity Enzymes Life Sciences Life Sciences & Biomedicine Membrane Biology Nanotechnology Neurobiology Science & Technology Substrate inhibition |
| title | A standard operating procedure for an enzymatic activity inhibition assay |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-14T19%3A46%3A04IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_webof&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=A%20standard%20operating%20procedure%20for%20an%20enzymatic%20activity%20inhibition%20assay&rft.jtitle=European%20biophysics%20journal&rft.au=Smirnovien%C4%97,%20Joana&rft.date=2021-05-01&rft.volume=50&rft.issue=3-4&rft.spage=345&rft.epage=352&rft.pages=345-352&rft.issn=0175-7571&rft.eissn=1432-1017&rft_id=info:doi/10.1007/s00249-021-01530-8&rft_dat=%3Cproquest_webof%3E2539544570%3C/proquest_webof%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2539544570&rft_id=info:pmid/33864100&rfr_iscdi=true |