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Quantifying Protein in 2-D PAGE Solubilization Buffers

  • Louis S. Ramagli
Part of the Methods in Molecular Biology book series (MIMB, volume 112)

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).

Keywords

Sodium Dodecyl Sulfate Solubilized Protein Concentration Colorectal Carcinoma Cell Line Carrier Ampholyte Basic Reagent 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc., Totowa, NJ 1999

Authors and Affiliations

  • Louis S. Ramagli
    • 1
  1. 1.Department of Molecular GeneticsM. D. Anderson Cancer CenterHouston

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