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Hindi Research Article
Optimal Concentration of 2,2,2-Trichloroacetic Acid for Protein Precipitation Based on Response Surface Methodology
Albert N Ngo, Miezan JM Ezoulin, Ibrahima Youm and Bi-Botti C Youan*Laboratory of Future Nanomedicines and Theoretical Chronopharmaceutics, Division of Pharmaceutical Sciences, University of Missouri-Kansas City, Kansas City, Mo 64108, USA
- *Corresponding Author:
- Bi-Botti C Youan
Laboratory of Future Nanomedicines and Theoretical Chronopharmaceutics
Division of Pharmaceutical Sciences
University of Missouri-Kansas City
Kansas City, Mo 64108, USA
Tel: +816 2352410
Fax: 816 235 5779
E-mail: youanb@umkc.edu
Received date: June 25, 2014; Accepted date: August 05, 2014; Published date: August 08, 2014
Citation: Ngo AN, Ezoulin MJM, Youm I, Youan BC (2014) Optimal Concentration of 2,2,2-Trichloroacetic Acid for Protein Precipitation Based on Response Surface Methodology. J Anal Bioanal Tech 5:198 doi: 10.4172/2155-9872.1000198
Copyright: 2014 Ngo AN, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
For low protein concentrations containing biological samples (in proteomics) and for non proteinaceous compound assays (in bioanalysis), there is a critical need for a simple, fast, and cost-effective protein enrichment or precipitation method. However, 2,2,2-trichloroacetic acid (TCA) is traditionally used for protein precipitation at ineffective concentrations for very low protein containing samples. It is hypothesized that response surface methodology, can be used to systematically identify the optimal TCA concentration for protein precipitation in a wider concentration range. To test this hypothesis, a central composite design is used to assess the effects of two factors (X1 = volume of aqueous solution of protein, and X2 = volume of TCA solution 6.1N) on the optical absorbance of the supernatant (Y1), and the percentage of protein precipitated (Y2). Using either bovine serum albumin (BSA) as a model protein or human urine (with 20 ppm protein content), 4% w/v (a saddle point) is the optimal concentration of the TCA solution for protein precipitation that is visualized by SDS-PAGE analysis. At this optimal concentration, the Y2-values range from 76.26 to 92.67% w/w for 0.016 to 2 mg/mL of BSA solution. It is also useful for protein enrichment and xenobiotic analysis in protein-free supernatant as applied to tenofovir (a model HIV microbicide). In these conditions, the limit of detection and limit of quantitation of tenofovir are respectively 0.0014 mg/mL and 0.0042 mg/mL. This optimal concentration of TCA provides optimal condition for protein purification and analysis of any xenobiotic compound like tenofovir.
