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.