ANALYSIS OF β-TUBULIN ISOFORMS BY ¹⁵N-LABELING AND MALDI-TOF MS
- CHANGE IN THEIR LEVELS IN BY2 CELLS THROUGH DIVISION -

Abstract

Tobacco BY2 cell components could be efficiently labeled with ¹⁵N by preculturing in ¹⁵N-substituted Nagata's medium. The cells labeled with stable isotope were mixed with the cells grown in a usual light medium, the acidic proteins were separated by 2-D PAGE, digested on membrane with AspN and analysed by MALDI-TOF MS. Accurate values of the change in the level of β-tubulin in 2-D spots through the period of division were obtained by measuring the ratio of signal intensities. As a result, no drastic changes as observed in the transcript levels took place for any isoform spots. However the spots did not necessarily consist of a single isotype product and moreover, the post-translational modifications, such as phosphorylation and glutamylation seemed to take place in some of them. The more efficient detection of acidic fragments are necessary and further investigations on this line is now under progress.