Optimization of chemiluminescent detection of mitochondrial RNA-protein interaction by nonradioactive mobility shift

  • Ahmed M El-Shehawi
  • Mona M Elseehy

Abstract

RNA mobility shift is one among many procedures used to study RNA-protein interaction. Yet, there are some limitations for the radioactive RNA mobility shift including; 1) the risk of using radiolabeled nucleotides, 2) the long time to get the results; this could range from days to weeks, and 3) its high cost as compared to nonradioactive techniques. In this study, we optimized a nonradioactive procedure using dig-11-UTP nucleotide and chemiluminescent detection for mitochondrial RNA-protein interaction. The optimizations include the quality limiting steps such as using non-specific competitors of RNA probe, UVcross linking time, electrotransfer to membranes, application with various protein extracts, and the examination for false positive RNA-protein complexes using proteinase K digestion. The results show that the optimizations carried out in the study significantly enhanced the quality of the results obtained with this procedure.

Keywords: RNA mobility shift, mitochondria, wheat, RNA-protein interaction, chemiluminescent detection, nonradioactive

African Journal of Biotechnology Vol. 12(16), pp. 1915-1920

Author Biographies

Ahmed M El-Shehawi
Department of Biotechnology, Faculty of Science, Taif University, Taif 21974, Saudi Arabia; Department of Genetics, Faculty of Agriculture, Alexandria University, Alexandria, Egypt
Mona M Elseehy
Department of Genetics, Faculty of Agriculture, Alexandria University, Alexandria, Egypt
Published
2016-01-28
Section
Articles

Journal Identifiers


eISSN: 1684-5315