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Determination of protein concentration in skeletal muscle using two spectrophotometric assays: the Lowry and the Bradford
Author(s) -
Seevaratnam Rajini,
Patel Barkha,
Hamadeh Mazen J
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.252
Subject(s) - lowry protein assay , bradford protein assay , bicinchoninic acid assay , chromatography , chemistry , skeletal muscle , reagent , biochemistry , biology , anatomy , casein
Background: Laboratory research often involves protein analysis, particularly for the comparison of protein expression and activity. Therefore, determination of protein concentration is an important first step prior to biochemical analyses. The Lowry and Bradford methods for protein determination are the most commonly used today, yet vary in several aspects. To date, no comparisons have been made in skeletal muscle tissue. Objective: We compared protein concentrations of mouse red and white gastrocnemius (N = 52), range of linearity, reagent stability and protein stability, using both the Lowry and Bradford assays. Results: Protein concentration determined by the Lowry (mean ± SD: 5.95 ± 1.45 mg/ml) was on average 15% higher than the Bradford (5.08 ± 1.31 mg/ml). We found a moderate correlation (r = 0.36, P = 0.01) with a slope of 0.39 ± 0.15 between the two methods. However, the Bland‐Altman test revealed a considerable bias (15.8 ± 29.7%; range: −42% to +74%). The linear range of concentration was smaller for the Lowry (0.05–0.50 mg/ml) than the Bradford (0–2.0 mg/ml). Lowry reagents were more stable over an hour than those of the Bradford (5.6% vs. 14.6% difference, respectively). Peak protein concentrations were reached immediately with the Lowry assay, and between 7–10 min with the Bradford assay. Conclusion: We have determined that although both the Lowry and Bradford assays measure protein concentration in skeletal muscle, the two methods are not interchangeable. Both methods have various strengths and weaknesses and should be considered before analysis. (This research was supported by the Hamilton Health Sciences Foundation and Faculty of Health‐York University).