Zymography: A Single-Step Staining Method for Quantitation of Proteolytic Activity on Substrate Gels
Zymography is an electrophoretic method for measuring proteolytic activity. The method is based on an sodium dodecyl sulfate gel impregnated with a protein substrate which is degraded by the proteases resolved during the incubation period. Coomassie blue staining of the gel reveals sites of proteoly...
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| Veröffentlicht in: | Analytical biochemistry 1997-06, Vol.249 (1), p.24-28 |
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| container_title | Analytical biochemistry |
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| creator | Leber, Thomas M. Balkwill, Frances R. |
| description | Zymography is an electrophoretic method for measuring proteolytic activity. The method is based on an sodium dodecyl sulfate gel impregnated with a protein substrate which is degraded by the proteases resolved during the incubation period. Coomassie blue staining of the gel reveals sites of proteolysis as white bands on a dark blue background. Within a certain range the band intensity can be related linearly to the amount of protease loaded. Although the method is widely used, crucial points concerning quantitation of proteolytic activity have not been rigorously addressed. In this report we describe a new staining protocol which converts the independent staining and destaining procedure into a single step. This leads to fast and reproducible staining of zymograms permitting reliable quantitation of proteolytic activity. As shown for proMMP-9 (type IV gelatinase-b) proteolytic activity can be quantitated in a linear manner in as little as 1 h of zymogram staining. We established the detection limit for proMMP-9 (32 pg), the linear range (below 1000 pg), and the reproducibility of the assay (coefficient of variation |
| doi_str_mv | 10.1006/abio.1997.2170 |
| format | Article |
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The method is based on an sodium dodecyl sulfate gel impregnated with a protein substrate which is degraded by the proteases resolved during the incubation period. Coomassie blue staining of the gel reveals sites of proteolysis as white bands on a dark blue background. Within a certain range the band intensity can be related linearly to the amount of protease loaded. Although the method is widely used, crucial points concerning quantitation of proteolytic activity have not been rigorously addressed. In this report we describe a new staining protocol which converts the independent staining and destaining procedure into a single step. This leads to fast and reproducible staining of zymograms permitting reliable quantitation of proteolytic activity. As shown for proMMP-9 (type IV gelatinase-b) proteolytic activity can be quantitated in a linear manner in as little as 1 h of zymogram staining. We established the detection limit for proMMP-9 (32 pg), the linear range (below 1000 pg), and the reproducibility of the assay (coefficient of variation <15%). This improved protocol is fast and reproducible. Its linear range of almost two log scales permits assay of proteolytic activity in a wider range than current methods.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1006/abio.1997.2170</identifier><identifier>PMID: 9193704</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Collagenases - analysis ; Electrophoresis, Polyacrylamide Gel ; Gels ; Linear Models ; Matrix Metalloproteinase 9 ; Reproducibility of Results ; Rosaniline Dyes ; Sensitivity and Specificity ; Staining and Labeling - methods</subject><ispartof>Analytical biochemistry, 1997-06, Vol.249 (1), p.24-28</ispartof><rights>1997 Academic Press</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c450t-24788c4cf96b2f533322a6c3a201813ce59ff0a366e7043173f076a46fd55d203</citedby><cites>FETCH-LOGICAL-c450t-24788c4cf96b2f533322a6c3a201813ce59ff0a366e7043173f076a46fd55d203</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0003269797921709$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/9193704$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Leber, Thomas M.</creatorcontrib><creatorcontrib>Balkwill, Frances R.</creatorcontrib><title>Zymography: A Single-Step Staining Method for Quantitation of Proteolytic Activity on Substrate Gels</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>Zymography is an electrophoretic method for measuring proteolytic activity. The method is based on an sodium dodecyl sulfate gel impregnated with a protein substrate which is degraded by the proteases resolved during the incubation period. Coomassie blue staining of the gel reveals sites of proteolysis as white bands on a dark blue background. Within a certain range the band intensity can be related linearly to the amount of protease loaded. Although the method is widely used, crucial points concerning quantitation of proteolytic activity have not been rigorously addressed. In this report we describe a new staining protocol which converts the independent staining and destaining procedure into a single step. This leads to fast and reproducible staining of zymograms permitting reliable quantitation of proteolytic activity. As shown for proMMP-9 (type IV gelatinase-b) proteolytic activity can be quantitated in a linear manner in as little as 1 h of zymogram staining. We established the detection limit for proMMP-9 (32 pg), the linear range (below 1000 pg), and the reproducibility of the assay (coefficient of variation <15%). This improved protocol is fast and reproducible. Its linear range of almost two log scales permits assay of proteolytic activity in a wider range than current methods.</description><subject>Collagenases - analysis</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Gels</subject><subject>Linear Models</subject><subject>Matrix Metalloproteinase 9</subject><subject>Reproducibility of Results</subject><subject>Rosaniline Dyes</subject><subject>Sensitivity and Specificity</subject><subject>Staining and Labeling - methods</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1997</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kM1LxDAQxYMoun5cvQk5ees6adq08baIX6CorF68hGw60Ui3WZNU6H9vl128OZdheG8eMz9CThlMGYC40Avnp0zKapqzCnbIhIEUGXCQu2QCADzLhawOyGGMXwCMFaXYJ_uSSV5BMSHN-7D0H0GvPodLOqNz1320mM0Trug8adeNM33E9Okban2gL73ukks6Od9Rb-lz8Al9OyRn6Mwk9-PSQEdp3i9iCjohvcU2HpM9q9uIJ9t-RN5url-v7rKHp9v7q9lDZooSUpYXVV2bwlgpFrktOed5roXhOgdWM26wlNaC5kLgeDtnFbdQCV0I25RlkwM_Iueb3FXw3z3GpJYuGmxb3aHvo6okiHqs0TjdGE3wMQa0ahXcUodBMVBrrGqNVa2xqjXWceFsm9wvltj82bccR73e6OO3-OMwqGgcdgYbF9Ak1Xj3X_Qv0GGGgw</recordid><startdate>19970615</startdate><enddate>19970615</enddate><creator>Leber, Thomas M.</creator><creator>Balkwill, Frances R.</creator><general>Elsevier Inc</general><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></search><sort><creationdate>19970615</creationdate><title>Zymography: A Single-Step Staining Method for Quantitation of Proteolytic Activity on Substrate Gels</title><author>Leber, Thomas M. ; Balkwill, Frances R.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c450t-24788c4cf96b2f533322a6c3a201813ce59ff0a366e7043173f076a46fd55d203</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1997</creationdate><topic>Collagenases - analysis</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Gels</topic><topic>Linear Models</topic><topic>Matrix Metalloproteinase 9</topic><topic>Reproducibility of Results</topic><topic>Rosaniline Dyes</topic><topic>Sensitivity and Specificity</topic><topic>Staining and Labeling - methods</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Leber, Thomas M.</creatorcontrib><creatorcontrib>Balkwill, Frances R.</creatorcontrib><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><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Leber, Thomas M.</au><au>Balkwill, Frances R.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Zymography: A Single-Step Staining Method for Quantitation of Proteolytic Activity on Substrate Gels</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>1997-06-15</date><risdate>1997</risdate><volume>249</volume><issue>1</issue><spage>24</spage><epage>28</epage><pages>24-28</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><abstract>Zymography is an electrophoretic method for measuring proteolytic activity. The method is based on an sodium dodecyl sulfate gel impregnated with a protein substrate which is degraded by the proteases resolved during the incubation period. Coomassie blue staining of the gel reveals sites of proteolysis as white bands on a dark blue background. Within a certain range the band intensity can be related linearly to the amount of protease loaded. Although the method is widely used, crucial points concerning quantitation of proteolytic activity have not been rigorously addressed. In this report we describe a new staining protocol which converts the independent staining and destaining procedure into a single step. This leads to fast and reproducible staining of zymograms permitting reliable quantitation of proteolytic activity. As shown for proMMP-9 (type IV gelatinase-b) proteolytic activity can be quantitated in a linear manner in as little as 1 h of zymogram staining. We established the detection limit for proMMP-9 (32 pg), the linear range (below 1000 pg), and the reproducibility of the assay (coefficient of variation <15%). This improved protocol is fast and reproducible. Its linear range of almost two log scales permits assay of proteolytic activity in a wider range than current methods.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>9193704</pmid><doi>10.1006/abio.1997.2170</doi><tpages>5</tpages></addata></record> |
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| language | eng |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Collagenases - analysis Electrophoresis, Polyacrylamide Gel Gels Linear Models Matrix Metalloproteinase 9 Reproducibility of Results Rosaniline Dyes Sensitivity and Specificity Staining and Labeling - methods |
| title | Zymography: A Single-Step Staining Method for Quantitation of Proteolytic Activity on Substrate Gels |
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