The answer depends on the question: Optimal conditions for western blot characterization of muscle collagen type 1 depends on desired isoform

Authors

  • Victoria J. Iannarone Lewis Katz School of Medicine, Temple University
  • Geneva E. Cruz Lewis Katz School of Medicine, Temple University
  • Brendan A. Hilliard Lewis Katz School of Medicine, Temple University
  • Mary F. Barbe Lewis Katz School of Medicine, Temple University

DOI:

https://doi.org/10.14440/jbm.2019.289

Keywords:

collagen type 1, muscle, mature collagen, procollagen, western blot

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.


Author Biographies

Victoria J. Iannarone, Lewis Katz School of Medicine, Temple University

Equal contributing first author. Medical student at Lewis Katz School of Medicine.

Geneva E. Cruz, Lewis Katz School of Medicine, Temple University

Equal contributing first author. Research Assistant in Department of Anatomy and Cell Biology.

Brendan A. Hilliard, Lewis Katz School of Medicine, Temple University

Senior Research Scientist in the Department of Anatomy and Cell Biology.

Mary F. Barbe, Lewis Katz School of Medicine, Temple University

Full Professor with tenure in the Department of Anatomy and Cell Biology.

References

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Published

2019-08-15

How to Cite

1.
Iannarone VJ, Cruz GE, Hilliard BA, Barbe MF. The answer depends on the question: Optimal conditions for western blot characterization of muscle collagen type 1 depends on desired isoform. J Biol Methods [Internet]. 2019Aug.15 [cited 2022May27];6(3):e117. Available from: https://jbmethods.org/jbm/article/view/289

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Protocols