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67. , Crawford, S. , Estes, M. , and Cohen, J. (1991) Expression of rotavirus VP2 produces empty corelike particles. J. Virol. 65, 2946–2952. 68. Crawford, S. , Burroughs, M. , Zhou, Y. , and Estes, M. K. (1994) Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells. J. Virol. 68, 5945–5952. 69. Shaw, A. , Samal, S. , and Prasad, B. V. (1996) Structure of aquareovirus shows how the different geometries of the two layers of the capsid are reconciled to provide symmetrical interactions and stabilization.

34: Rotaviruses: Methods and Protocols Edited by: J. Gray and U. , Totowa, NJ 33 34 Patton et al. propagation. MA104 cells can be grown to confluency in either minimum essential medium or Medium 199 containing 5% fetal bovine serum (FBS). However, in this laboratory, higher virus yields are obtained with the latter. 2. Preparation of Virions Containing Uncleaved Virion Protein VP4 The presence of trypsin in the medium during RV propagation will result in the cleavage of the virion protein VP4 into VP5* (60kDa) and VP8* (27kDa) (2) and, thus, virus purified from trypsin-containing media will contain little if any intact VP4.

2. ASSESSING STABILITY OF PROBE–PROTEIN INTERACTIONS The strength of the interaction between an RNA and a protein can be evaluated by monitoring the effect of including different concentrations of salt in the gel-shift assay. 3. DISTINGUISHING BETWEEN SPECIFIC AND NONSPECIFIC RNA-BINDING ACTIVITIES Because RNA-binding proteins generally display some degree of nonspecific affinity for single-stranded RNA, competitive gel-shift assays should be performed to determine whether the RNA-binding activity observed for any protein is specific or nonspecific (21).

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A Characterization of Afline Cylinders by Opozda B.


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