Preparation of polyclonal antibody specific for AtPLC4, an Arabidopsis phosphatidylinositol-specific phospholipase C in rabbits

Phosphoinositide-specific phospholipase Cs (PI-PLCs) are important enzymes in eukaryotes, which catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol 1,4,5-trisphosphate and diacylglycerol. The Arabidopsis genome contains nine putative PI-PLC genes. AtPLC4, an abiotic stress induced gene, has been reported to encode an active PI-PLC isoform. However, the exact roles of putative AtPLC4 in plant remain to be elicited. The first 108 amino acid residues of the N-terminal of AtPLC4, referred to as AtPLC4 N, was expressed as a recombinant protein in Escherichia coli and used as antigen in generating antibody. Purified recombinant proteins including AtPLC1 to AtPLC5, AtPLC8, AtPLC9 and AtPLC4 N were transferred onto the same blot to test specificity of the prepared antibody. Western blot result shows that only AtPLC4 and AtPLC4 N can be recognized by the antibody. The antibody recognized a protein of approximately 68kDa in the plasma membrane fraction and cytosolic fractions prepared from Arabidopsis thaliana plants. This corresponds very well with the calculated molecular weight of AtPLC4. The results suggest that AtPLC4 may encode a plasma membrane-associated protein.