Abstract
Accurate quantitative and qualitative comparison of resolved proteins by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) requires that identical amounts of sample be loaded on each gel. Use of trichloroacetic acid (TCA) or organic solvent precipitation protocols followed by a protein assay results in either over- or underestimation of solubilized protein concentration. Furthermore, the practice of loading equivalent amounts of radioactively labeled protein-based counts/min or loading extract from the same number of cells is inaccurate. The author has observed that radioactively labeled cell extracts exhibit 10–20% variances in protein concentration. Similarly, solubilization of 2×106 cells from a number of colorectal carcinoma cell lines resulted in protein concentrations ranging from 1.60 to 3.93 μg protein/μL buffer (1). Therefore, the ability to quantify protein actually solubilized in 2-D PAGE sample buffers is necessary to allow users to adjust for variable solubility properties of protein(s).
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References
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© 1999 Humana Press Inc., Totowa, NJ
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Ramagli, L.S. (1999). Quantifying Protein in 2-D PAGE Solubilization Buffers. In: Link, A.J. (eds) 2-D Proteome Analysis Protocols. Methods in Molecular Biology, vol 112. Humana Press. https://doi.org/10.1385/1-59259-584-7:99
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DOI: https://doi.org/10.1385/1-59259-584-7:99
Publisher Name: Humana Press
Print ISBN: 978-0-89603-524-9
Online ISBN: 978-1-59259-584-6
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