Essential modification of the Sircol Collagen Assay for the accurate quantification of collagen content in complex protein solutions
Collagen contains the unique imino acid hydroxyproline (HyPro), which is involved in the stabilization of this triple helical molecule. The concentration of HyPro is customarily used to calculate the total collagen content in a cell culture environment and in acid hydrolysates of normal and pathophy...
| Main Authors: | , , , , |
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| Format: | Journal Article |
| Published: |
Elsevier BV
2010
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| Online Access: | http://hdl.handle.net/20.500.11937/18496 |
| _version_ | 1848749760273448960 |
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| author | Lareu, Ricky R. Zeugolis, D. Abu-Rub, M. Pandit, A. Raghunath, M. |
| author_facet | Lareu, Ricky R. Zeugolis, D. Abu-Rub, M. Pandit, A. Raghunath, M. |
| author_sort | Lareu, Ricky R. |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | Collagen contains the unique imino acid hydroxyproline (HyPro), which is involved in the stabilization of this triple helical molecule. The concentration of HyPro is customarily used to calculate the total collagen content in a cell culture environment and in acid hydrolysates of normal and pathophysiological tissues. Radiolabelling, chromatographic and calorimetric assays have been developed over the years for the accurate determination of collagen content through HyPro estimation. Recently, the Sircol Collagen Assay (SCA) has been almost exclusively adopted as the fastest and simplest colorimetric method for the determination of collagen concentration in complex protein solutions. We show here that the colorimetric SCA, which is based on the binding of Sirius red (SR) to collagen, is flawed by interference of non-collagenous proteins (e.g. serum). In fact, we demonstrate that SCA in cell culture systems and tissue hydrolysates results in a dramatic overestimation of collagen content ranging from 3- to 24-fold. In order to rescue this otherwise very practical assay, we introduce a simple purification procedure that allows the removal of interfering non-collagenous proteins from culture media and tissue samples so that accurate measurements with SCA are now possible. |
| first_indexed | 2025-11-14T07:26:03Z |
| format | Journal Article |
| id | curtin-20.500.11937-18496 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| last_indexed | 2025-11-14T07:26:03Z |
| publishDate | 2010 |
| publisher | Elsevier BV |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-184962017-09-13T13:43:52Z Essential modification of the Sircol Collagen Assay for the accurate quantification of collagen content in complex protein solutions Lareu, Ricky R. Zeugolis, D. Abu-Rub, M. Pandit, A. Raghunath, M. Ultrafiltration Hydroxyproline assay Collagen content Sircol assay Sirius red Collagen contains the unique imino acid hydroxyproline (HyPro), which is involved in the stabilization of this triple helical molecule. The concentration of HyPro is customarily used to calculate the total collagen content in a cell culture environment and in acid hydrolysates of normal and pathophysiological tissues. Radiolabelling, chromatographic and calorimetric assays have been developed over the years for the accurate determination of collagen content through HyPro estimation. Recently, the Sircol Collagen Assay (SCA) has been almost exclusively adopted as the fastest and simplest colorimetric method for the determination of collagen concentration in complex protein solutions. We show here that the colorimetric SCA, which is based on the binding of Sirius red (SR) to collagen, is flawed by interference of non-collagenous proteins (e.g. serum). In fact, we demonstrate that SCA in cell culture systems and tissue hydrolysates results in a dramatic overestimation of collagen content ranging from 3- to 24-fold. In order to rescue this otherwise very practical assay, we introduce a simple purification procedure that allows the removal of interfering non-collagenous proteins from culture media and tissue samples so that accurate measurements with SCA are now possible. 2010 Journal Article http://hdl.handle.net/20.500.11937/18496 10.1016/j.actbio.2010.02.004 Elsevier BV restricted |
| spellingShingle | Ultrafiltration Hydroxyproline assay Collagen content Sircol assay Sirius red Lareu, Ricky R. Zeugolis, D. Abu-Rub, M. Pandit, A. Raghunath, M. Essential modification of the Sircol Collagen Assay for the accurate quantification of collagen content in complex protein solutions |
| title | Essential modification of the Sircol Collagen Assay for the accurate quantification of collagen content in complex protein solutions |
| title_full | Essential modification of the Sircol Collagen Assay for the accurate quantification of collagen content in complex protein solutions |
| title_fullStr | Essential modification of the Sircol Collagen Assay for the accurate quantification of collagen content in complex protein solutions |
| title_full_unstemmed | Essential modification of the Sircol Collagen Assay for the accurate quantification of collagen content in complex protein solutions |
| title_short | Essential modification of the Sircol Collagen Assay for the accurate quantification of collagen content in complex protein solutions |
| title_sort | essential modification of the sircol collagen assay for the accurate quantification of collagen content in complex protein solutions |
| topic | Ultrafiltration Hydroxyproline assay Collagen content Sircol assay Sirius red |
| url | http://hdl.handle.net/20.500.11937/18496 |