Fig. 5.

Non-denaturing PAGE and co-immunopurification of castor PEPC isozymes. (A) Non-denaturing PAGE followed by in-gel PEPC activity staining and immunoblotting was performed on clarified extracts from stage VII (full cotyledon) developing COS endosperm and cotyledon (5 mU per lane), alongside ammonium sulphate-concentrated extracts from the inner integument of stage I (proembryo) COS (10 mU per lane) and 5 d germinating roots (3 mU per lane). Non-denaturing PAGE was also performed on PEPC from expanding castor leaves which had been partially purified by ammonium sulphate fractionation and Butyl-Sepharose hydrophobic chromatography (15 mU PEPC for activity stain and anti-BTPC, 5 mU PEPC activity for anti-PTPC). (B, C) A clarified extract originating from 10 g of the inner integument was subjected to immunopurification using 2 ml anti-PTPC (B) or anti-BTPC (C) immunoaffinity columns. After washing non-absorbed proteins with P_i-buffered saline, the bound proteins were eluted using 100 mM glycine-HCl (pH 2.8), neutralized, and concentrated. The initial extract (Extract; 5 μg per lane), concentrated flow through fractions (Co-IP FT; 5 μg per lane), and concentrated eluates (Co-IP; 5 μg for anti-PTPC, 1 μg for anti-BTPC) were subjected to SDS-PAGE followed by immunoblotting with anti-PTPC and anti-BTPC.