Purification and characterization of U small nuclear ribonucleoproteins in cesium chloride gradients

Abstract

This chapter describes a protocol based on the stability of U snRNPs in high salt concentrations, allowing their purification by isopycnic centrifugation in cesium chloride gradients. Any cells growing in suspension culture or obtained from ascitic fluid, as well as tissues, can be used as starting material. The disrupted cells are immediately layered on top of four 10-ml cushions of Buffer 3 and centrifuged for 20 min at 3,000 g. Material that does not enter the sucrose layer (cytoplasmic fraction) is discarded. The pellets of nuclei are washed twice with 5-10 ml of Buffer 4. The most satisfactory procedure is to adsorb snRNPs onto a DEAE-Sepharose column from which they are step-eluted. The nucleoplasmic extract is applied to a 5-ml DEAE-Sepharose CL-6B (Pharmacia) column (0.8 cm diameter) in equilibration buffer. When the isolated particles are to be used in splicing systems, they are dialyzed against the suitable buffer is and concentrated by centrifugation in Centricon 10 microconcentrators (Amicon), to a concentration estimated to be very similar to that of the corresponding snRNP in a splicing nuclear extract. 2] Structural Analysis of U Small Nuclear