A simple and efficient method for processing of cell lysates for two-dimensional gel electrophoresis
Publication Type:Journal Article
Source:Electrophoresis, Volume 31, Number 14, p.2429-2435 (2010)
Keywords:acetone, ammonium acetate, article, Cell Line, cell lysate, cell protein, Chemical precipitation, chloroform, controlled study, Electrophoresis, enzyme activity, enzyme denaturation, Ethanol, gel, human, human tissue, Humans, isopentyl alcohol, methanol, phenol, precipitation, protein analysis, proteinase, Proteins, proteomics, qualitative analysis, solubilization, thiourea, two dimensional gel electrophoresis, Two-Dimensional, ultrasound, urea
Sample preparation is one of the major issues in 2-DE for the separation of proteins. Although a 100% representation of cellular proteins onto a 2-DE is virtually impossible, maximum representation of cellular proteins compared with the original cell lysate is important in the subsequent analysis. We demonstrate that lysis of cells in urea/thiourea solution with subsequent sonication to disrupt the nucleic acids and concentration of the lysate using centri-con led to enrichment of proteins. The procedure resulted in minimal nucleic acid contamination with better resolution of spots. 2-DE spot patterns of proteins prepared using urea - thiourea solubilization/centri-con method to other protein enrichment methods such as phenol/chloroform/isoamyl alcohol extraction, methanol/ammonium acetate precipitation, acetone precipitation and ethanol precipitation were compared. Urea - thiourea solubilization combined with centri-con method of protein enrichment represented higher number/unique spots particularly in the 50-250 kDa M r compared with others. Lysis of cells in urea/thiourea from the beginning of lysate preparation preserves the proteins from protease activity due to denaturation of proteases. Thus, we demonstrate that the centri-con methodology is simple and effective for the preparation of high-quality sample that can be used for a qualitative representation of cellular proteins on a 2-DE for proteomic analysis. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.
cited By (since 1996)2
Cite this Research Publication
Related Research Publications
- Neuroproteomics: Are we biased in our representation of molecular targets associated with specific domains? Implications in biomarker discovery
- A novel unbiased proteomic approach to detect the reactivity of cerebrospinal fluid in neurological diseases
- Co-purified gelatinases alter the stability and biological activities of human plasma fibronectin preparations
- Metabolism of proteins and glycoproteins in tumour bearing mice treated with Aeromonas L-asparaginase.
- Proteogenomics of Candida tropicalis- An Opportunistic Pathogen with Importance for Global Health