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Purification and Electron Microscopy of Spliceosomal snRNPs

  • Berthold Kastner
Part of the Springer Lab Manual book series (SLM)

Abstract

The main focus of this chapter is electron microscopy (EM) of RNP complexes using the negative staining technique. This technique is fast and relatively easy to perform and is adequate for imaging with a standard transmission electron microscope. The prerequisite for EM analysis is the availability of isolated intact RNP particles with a concentration not less than 10–20 μg/ml. The lower size limit of the particles that can be studied by the method depends very much on their actual shape. This chapter deals with the spliceosomal RNP subunits, called small nuclear ribonucleoproteins (snRNPs). Because of their sizes, which range from about 300 kDa (U1snRNP) to up to 1500 kDa (U4/U6.U5 tri-snRNP), the snRNPs are ideal objects for EM analysis. However, snRNP protein sub complexes as small as ~60 kDa have also been investigated successfully by negative staining EM. EM analysis is greatly facilitated if the particles studied are available as highly purified samples. Therefore, isolation procedures for snRNPs are described here in some detail.

Keywords

Carbon Film Ribosomal Subunit Sucrose Gradient Centrifugation Small Nuclear Ribonucleoprotein Immune Electron Microscopy 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag Berlin Heidelberg 1998

Authors and Affiliations

  • Berthold Kastner
    • 1
  1. 1.Institute of Molecular Biology and Tumor ResearchPhilipps UniversitätMarburgGermany

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