Sample pooling in 2-D gel electrophoresis: a new approach to reduce nonspecific expression background.
Protein expression alterations unrelated to an investigated phenotype are accumulated in most cell line models during establishment. Performing a whole proteome screening of lymphoma cell lines, we established a method to reduce the influence of protein expression unrelated to the distinct investigated phenotype. In 2-D PAGE, the comprehensive analysis of a large number of protein spots would be simplified by pooling cell line samples of the investigated phenotype. Applying this pooling approach, unrelated alterations of single samples are 'muted' by dilution. Analysing two different lymphoma subtypes (follicular and mantle cell lymphoma) by this method, spots originating in only single cell lines were reduced by 72% (650/900), whereas even modestly altered expression of protein spots detected in all lines were reliably detected in the pooled protein gels. We conclude that our pooling approach is a preferable approach to reliably detect a common protein expression pattern and may even allow proteomic analysis of clinical samples with limited amounts of sample material, even with minimal cell numbers as low as 1 x 10(6).
Weinkauf
Marc
M
Department of Medicine III, University Hospital Grosshadern/LMU, Munich, Germany.
Hiddemann
Wolfgang
W
Dreyling
Martin
M
D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D008224:Lymphoma, Follicular / Q000737:chemistry / Q000235:genetics*; D020522:Lymphoma, Mantle-Cell / Q000737:chemistry / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D040901:Proteomics / Q000379:methods*