3.5 Molecular weight estimation of CRCT
Western blot analysis was carried out to detect CRCT in the leaf sheath
of CRCT overexpression line and knockout line (Figure 6a). The
expression of CRCT was significantly increased in the CRCT
overexpression line. In contrast, the CRCT band was not detected in the
CRCT knockout line, which was produced by the CRISPR/Cas9 system.
To estimate the molecular weight of CRCT in vivo , gel filtration
chromatography was performed using the crude extract prepared from the
CRCT overexpression line. CRCT was eluted around fraction 22 (Figure
6b). Based on the relationship between the molecular weights of standard
proteins and their fraction numbers, the molecular weight of CRCT was
estimated to be 270 kDa (Figure 6c). According to the deduced amino acid
sequence of CRCT, a monomer of CRCT is only 34.2 kDa, suggesting that
CRCT may form oligomers or stably interact with other proteins.