A simple and efficient method for processing of cell lysates for two-dimensional gel electrophoresis.
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.
Xavier
Tessy
T
Division of Molecular Medicine, Amrita Research Institute, Amrita Institute for Medical Sciences, Ponekkara, Kochi, India.
Ganesan
Trivadi S
TS
Menon
Krishnakumar N
KN
D000096:Acetone / Q000737:chemistry; D002460:Cell Line; D011232:Chemical Precipitation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000191:economics / Q000379:methods*; D000431:Ethanol / Q000737:chemistry; D006801:Humans; D011506:Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000191:economics / Q000379:methods*; D013890:Thiourea / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D011506:Proteins; D000096:Acetone; D000431:Ethanol; D014508:Urea; D013890:Thiourea