Analysis of the Effect of Elevated Cytokinin Content on the Photosynthetic Apparatus Using Blue Native PAGE

Abstract

Blue Native gel electrophoresis is a very powerful technique for the separation of highly hydrophobic membrane proteins and the isolation of native protein complexes. It offers an alternative for two-dimensional gel electrophoresis. To study the effects of cytokinins on the composition of the complexes in the photosynthetic apparatus, transgenic Pssu-ipt tobacco plants with increased cytokinin content are used and compared to wild-type plants. After isolation of intact chloroplasts of transgenic and wild-type plants, digitonin and dodecyl-β-maltoside (1%) are used to obtain the native conformation of the photosynthetic complexes. A separation in the first dimension under native conditions, Blue Native PAGE, is combined with SDS-PAGE for separation of the protein complexes into their subunits. This provides a better insight in the network of protein complexes of the photosynthetic apparatus and the physiological changes due to elevated cytokinin content.