POL Scientific / JBM / Volume 6 / Issue 3 / DOI: 10.14440/jbm.2019.289
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The answer depends on the question: Optimal conditions for western blot characterization of muscle collagen type 1 depends on desired isoform

Victoria J. Iannarone1 Geneva E. Cruz1 Brendan A. Hilliard1 Mary F. Barbe1
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1 Department of Anatomy and Cell Biology, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, United States
Published: 15 August 2019
© 2019 by the author. Licensee POL Scientific, USA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
Abstract

Fibrillar collagen type 1 is the most abundant type of collagen within the body and is a critical component of extracellular infrastructure. In order to assess collagen synthesis and extracellular accumulation in fibrotic disorders, improved methods are needed to detect changes in procollagen versus mature collagen at the protein level. Using Western blot methodology, we systematically examined: (1) gel composition (Tris-glycine vs. bis-Tris, gradient vs. non-gradient, sodium dodecyl sulfate (SDS) vs. no SDS); (2) sample preparation (SDS vs. no SDS, β-mercaptoethanol (BME) vs. no BME, boiling vs. no boiling); and (3) running buffer composition (SDS vs. no SDS). Our results indicate full native gel conditions prevent resolution of all collagen type 1 bands. The best resolution of type 1 procollagens is achieved using 4%–12% Tris-glycine gels without the presence of SDS in the gel itself, although SDS in the running and sample buffers are needed. Also, BME must not be added to the sample buffer and samples should not be boiled. For characterization of mature collagen 1(I), both 8% and gradients type gels are appropriate, although still without SDS, yet with SDS included in both running and sample buffers, BME must be added to the sample buffer, and samples should not be boiled. Boiling is to be avoided as the antigenic site recognized by the monoclonal antibody used is sensitive to thermal denaturation, as is the case with many monoclonal antibodies available on the market. Thus, the exact parameters employed are dependent upon the collagen protein product that the scientist desires to identify.

Keywords
collagen type 1
muscle
mature collagen
procollagen
western blot
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Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TBA, Published by POL Scientific