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Open Access Highly Accessed Research article

Variability of protein level and phosphorylation status caused by biopsy protocol design in human skeletal muscle analyses

Marc-André Caron1, Steve J Charette12, François Maltais1 and Richard Debigaré1*

Author Affiliations

1 Centre de recherche, Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Québec, Canada

2 Institut de biologie intégrative et des systèmes, Pavillon Charles-Eugène-Marchand, Université Laval, Québec Canada

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BMC Research Notes 2011, 4:488  doi:10.1186/1756-0500-4-488

Published: 10 November 2011

Abstract

Background

Bergström needle biopsy is widely used to sample skeletal muscle in order to study cell signaling directly in human tissue. Consequences of the biopsy protocol design on muscle protein quantity and quality remain unclear. The aim of the present study was to assess the impact of different events surrounding biopsy protocol on the stability of the Western blot signal of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), Akt, glycogen synthase kinase-3β (GSK-3β), muscle RING finger protein 1 (MuRF1) and p70 S6 kinase (p70 S6K). Six healthy subjects underwent four biopsies of the vastus lateralis, distributed into two distinct visits spaced by 48 hrs. At visit 1, a basal biopsy in the right leg was performed in the morning (R1) followed by a second in the left leg in the afternoon (AF). At visit 2, a second basal biopsy (R2) was collected from the right leg. Low intensity mobilization (3 × 20 right leg extensions) was performed and a final biopsy (Mob) was collected using the same incision site as R2.

Results

Akt and p70 S6K phosphorylation levels were increased by 83% when AF biopsy was compared to R1. Mob condition induced important phosphorylation of p70 S6K when compared to R2. Comparison of R1 and R2 biopsies revealed a relative stability of the signal for both total and phosphorylated proteins.

Conclusions

This study highlights the importance to standardize muscle biopsy protocols in order to minimize the method-induced variation when analyzing Western blot signals.