A simple and efficient method for processing of cell lysates for two-dimensional gel electrophoresis
Xavier, T; Ganesan, TS; Menon, KN
| HERO ID | 646346 |
|---|---|
| In Press | No |
| Year | 2010 |
| Title | A simple and efficient method for processing of cell lysates for two-dimensional gel electrophoresis |
| Authors | Xavier, T; Ganesan, TS; Menon, KN |
| Journal | Electrophoresis |
| Volume | 31 |
| Issue | 14 |
| Page Numbers | 2429-2435 |
| Abstract | 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. |
| Doi | 10.1002/elps.20090064 |
| Pmid | 20564265 |
| Wosid | WOS:000280709700018 |
| Is Certified Translation | No |
| Dupe Override | No |
| Comments | Source: Web of Science 000280709700018 |
| Is Public | Yes |
| Language Text | English |
| Keyword | 2-DE; Cell lysate; Centri-con; Sample preparation |
| Is Qa | No |