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Biacore’s surface plasmon resonance technology is widely used in the study of affinity and kinetics of interactions between two or more biomolecules, receptor-ligand interactions, and in understanding the kinetics of biomaterials. The analyte protein bound to the immobilized ligand on the sensor chip can be recovered for subsequent identification by mass spectrometry. Earlier studies have demonstrated that Biacore can be used to fish out proteins from crude mixtures in a highly purified state, and the recovered protein can be identified by upstream mass spectrometry. However, earlier studies have been done mainly for the known target protein. The study of RNA-binding proteins is very important for understanding the assembly and function of RNA-protein complexes in the splice-osome. In the current study, a biotinylated short single hairpin loop RNA was immobilized on the streptavidin sensor chip, and the HeLa cell nuclear extract was passed over it to identify the proteins bound to the biotinylated RNA stem-loop. The results of recovered analytes identified by mass spectrometry show that the Biacore 3000 could be a useful technique to identify previously unknown RNA-binding protein/s from nuclear extracts.