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A simple dot-blot–Sirius red-based assay for collagen quantification

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Abstract

The assessment of collagen content in tissues is important in biomedical research, since this protein is altered in numerous diseases. Hydroxyproline and Sirius red based assays are the most common methods for collagen quantification. However, these procedures have some pitfalls, such as the requirement of oxygen-free medium or expensive equipment and large sample size or being unsuitable for hydrolyzed collagen, respectively. Our objective was to develop a specific, versatile, and user-friendly quantitative method applicable to small tissue samples and extracts obtained from elastin purification, therefore, suitable for simultaneous quantification of elastin. This method is based on the binding of Sirius red to collagen present in a sample immobilized on a PVDF membrane, as in the dot-blot technique, and quantified by a scanner and image analysis software. Sample loading, Sirius red concentration, temperature and incubation time, type of standard substance, albumin interference, and quantification time are optimized. The method enabled the quantification of (1) intact collagen in several rat tissue homogenates, including small resistance-sized arteries, (2) partially hydrolyzed collagen obtained from NaOH extracts, compatible with elastin purification, and (3) for the detection of differences in collagen content between hypertensive and normotensive rats. We conclude that the developed technique can be widely used since it is versatile (quantifies intact and hydrolyzed collagen), requires small sample volumes, is user-friendly (low-cost, easy to use, minimum toxic materials, and reduced time of test), and is specific (minimal interference with serum albumin).

Dot-blot–Sirius red-based assay for collagen quantification

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Acknowledgment

This work was supported by the Ministerio de Educación y Ciencia from Spain (FEM2009-13434 C02-02 and FEM2012-37634-C03-01).

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  1. Luis Condezo-Hoyos

    Present address: School of Food Science and Human Nutrition, Washington State University, P.O. Box 646376, WA, Pullman, 99164-6376, USA

Authors and Affiliations

  1. Departamento de Fisiología, Facultad de Medicina, Universidad Autónoma de Madrid, Calle del Arzobispo Morcillo 4, 28029, Madrid, Spain

    Pilar Rodríguez-Rodríguez, Silvia M. Arribas, Angel Luis López de Pablo, M. Carmen González, Fatima Abderrahim & Luis Condezo-Hoyos

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  1. Pilar Rodríguez-Rodríguez
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  2. Silvia M. Arribas
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  3. Angel Luis López de Pablo
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  4. M. Carmen González
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  5. Fatima Abderrahim
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Correspondence to Luis Condezo-Hoyos.

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Rodríguez-Rodríguez, P., Arribas, S.M., de Pablo, A.L.L. et al. A simple dot-blot–Sirius red-based assay for collagen quantification. Anal Bioanal Chem 405, 6863–6871 (2013). https://doi.org/10.1007/s00216-013-7101-0

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  • DOI: https://doi.org/10.1007/s00216-013-7101-0

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