2-DE related techniques have been broadly used for protein separation and identification in recent years. However, many important cellular components often escape detection by 2-DE due to its inherent low resolution. Here we use a polyU-affinity column to enrich proteins with nucleotide-binding motifs. As a result, 26 enriched protein spots, including three new Arabidopsis RNA-binding proteins cp31A, cp31B, and CSP41, are identified by MALDI-TOF-MS. In the sum, 15 protein spots encoded by 12 individual genes are observed on the 2-DE only after binding to the polyU-affinity column. Two of these genes are expressed at lower than 0.1% the level of TUB4 that were considered mainly as non-expressed genes in several ATH1 microarray analysis of Arabidopsis leaf samples. Our results suggest that polyU or similar media may be combined with 2-DE techniques to identify cellular proteins with extremely low transcription levels to fulfill specific research needs.