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A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry
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A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Published on: March 28, 2017

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Technical note: Protocol for electrophoretic separation of bovine myosin heavy chain isoforms and comparison to

Tracy L Scheffler1, Megan B Leitner1, Shelby A Wright1

  • 1Department of Animal Sciences, University of Florida, Gainesville, FL.

Journal of Animal Science
|July 23, 2018
PubMed

Insights

A new sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method effectively separates bovine myosin heavy chain (MyHC) isoforms. This rapid SDS-PAGE technique offers a reliable alternative to traditional, time-consuming immunohistochemistry analysis.

Area of Science:

  • Muscle physiology
  • Biochemistry
  • Animal science

Background:

  • Myosin heavy chain (MyHC) isoform composition dictates muscle fiber contractile speed.
  • Current methods for bovine MyHC isoform analysis, such as immunohistochemistry, are time-consuming.
  • A validated, rapid electrophoretic method for bovine MyHC isoform separation is needed.

Purpose of the Study:

  • To develop and validate a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) procedure for separating bovine MyHC isoforms (I, IIa, IIx).
  • To compare the results of the developed SDS-PAGE method with immunohistochemistry (IHC) analysis.

Main Methods:

  • Muscle samples were collected from various bovine muscles within 1.5 hours postmortem.
  • Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was optimized by varying gel composition, specifically glycerol content.
  • Myosin heavy chain (MyHC) isoform identity was confirmed using western blotting.

Main Results:

  • Increasing glycerol content in polyacrylamide gels to 37% enabled clear separation of bovine MyHC isoforms.
  • MyHC isoform composition determined by SDS-PAGE showed consistency with IHC analysis (P > 0.2).

Conclusions:

  • Optimized SDS-PAGE provides clear separation of bovine MyHC isoforms.
  • This validated SDS-PAGE method is a viable and faster alternative to IHC for analyzing MyHC isoform composition in cattle.

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