Abstract
Serum proteome analysis provides a potential promising approach in disease diagnosis and therapeutic monitoring. However, the large dynamic range of proteins, high-abundant proteins, excess of salt and lipid in serum makes the analysis very challenging. Therefore, it is imperative to improve the dissolution of proteins in twodimensional gel electrophoresis (2-DE), and enhance the ability to analyze the proteome of serum under a wide variety of physiological conditions. This study examined the effects of various combinations of depletion highabundant protein, precipitated means, hot SDS treatment and IPG strips with different pH on 2-DE map for mouse serum. Finally the removal of high-abundant proteins in serum by the ProteoExtractTM Albumin Removal column, ethanol precipitation, the heating with 2.5% SDS and 2.3% DTT to denature sample at 95oC for 3 min, and IEF on pH 4-7 IPG strips (17cm) with 100 ?g depleted serum proteins are generally recommended for serum proteome analysis on 2-DE by silver staining, which can effectively improve the resolution and intensity of low-abundant proteins. The optimized conditions help to produce a better reference 2-DE gel of serum samples for following identification potential novel disease markers.
Highlights
Serum proteins are useful diagnostic tools and alteration of the expression of some serum proteins is an early sign of an altered physiology that may be indicative of disease (Poon and Johnson, 2001), serum is usually a source for biomarker discovery by proteomics analysis
Proteins were separated by isoelectric focusing (IEF) with precast IPG strips
200ug total proteins was diluted to 125ul with a sample rehydration buffer to run IEF
Summary
Serum proteins are useful diagnostic tools and alteration of the expression of some serum proteins is an early sign of an altered physiology that may be indicative of disease (Poon and Johnson, 2001), serum is usually a source for biomarker discovery by proteomics analysis. The large dynamic range of proteins in serum makes the analysis very challenging because high-abundant proteins, such as albumin, IgG, antitrypsin, etc, tend to mask those of lower abundance on two-dimensional gel electrophoresis (2-DE) (Ahmed et al, 2003). Many factors including sample preparation method, pH value of IPG stripe, staining approach, etc can affect protein separation and identification on 2-DE. 2-DE has major advantages in discovery of the overall alternations of specific proteins expression from serum sample (Anderson and Anderson, 1977; Omenn, 2004) , and the comparison of protein expression between normal and abnormal states of serum is widely applied to clinical and biomedical research in many reports (Judith et al, 2007; John et al, 2007; Bijon and Jürgen, 2007; Baukjede et al, 2008; Zhao et al, 2007; Feng et al, 2005).
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