This chapter discusses biochemical approaches that can be used to isolate, purify, and characterize RNA-binding proteins in Drosophila melanogaster. The same procedures are also applicable to the isolation of proteins from other tissues, other organisms, and from cultured cells. The most common affinity chromatography procedure used for the isolation of RNA-binding proteins from D. melanogaster is single stranded (ssDNA)-cellulose chromatography. Overall, purification can be accomplished rapidly and on a large scale. It does not depend on the integrity of RNA and does not require protein denaturing conditions. Individual RNA-binding proteins can be separated from one another on the same column, owing to their different affinities for ssDNA. This procedure is used to isolate and characterize heterogeneous nuclear ribonucleoprotein (hnRNP) proteins from human HeLa cells. Proteins not related to RNA metabolism may also bind to ssDNA-cellulose and conversely, not all RNAbinding proteins will necessarily bind to ssDNA. Therefore, binding to ssDNA is not by itself an absolute diagnostic criterion for RNA-binding proteins, and the identification of genuine RNA-binding proteins must be supported by complimentary data.
ASJC Scopus subject areas
- Cell Biology