Isoelectric point-based prefractionation of proteins from crude biological samples prior to two-dimensional gel electrophoresis.
Two-dimensional gel electrophoresis (2-DE) is used to compare the protein profiles of different crude biological samples. Narrow pH range Immobilized pH Gradient (IPG) strips were designed to increase the resolution of these separations. To take full advantage of IPG strips, the ideal sample should be composed primarily of proteins that have isoelectric point (pI) values within the pH range of the IPG strip. Prefractionation of cell lysates from a human prostate cancer cell line cultured in the presence or absence of epigallocatechin-3-gallate was achieved in fewer than 30 min using an anion-exchange resin and two expressly designed buffers. The procedure was carried out in a centrifuge tube and standard instrumentation was used. The cell lysates were prefractionated into two fractions: proteins with pI values above 7 and between 4 and 7, respectively. The fractions were then analyzed by 2-DE, selecting appropriate pH ranges for the IPG strips, and the gels were compared with those of unprefractionated cell lysates. Protein loading capacity was optimized and resolution and visualization of the less abundant and differentially expressed proteins were greatly improved.
Sahab
Ziad J
ZJ
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306-4390, USA.
Suh
Yewseok
Y
Sang
Qing-Xiang Amy
QX
D045744:Cell Line, Tumor; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D008297:Male; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology; D018892:Proton-Motive Force; D016622:Silver Staining