Production and purification of polyclonal antibodies to Pisum and Avena labile phytochrome which cross-react with phyA from Pharbitis nil

Little work was done so far with phytochrome from Pharbitis nil. Purification of phyA from this plant has been exceptionally difficult. Labile phytochrome was presented in too small amount to obtain either absorption spectra or enough protein to produce antibodies. Monoclonal antibodies mAP5, MAC 50, 52, 198 recognized Pharbitis nil labile phytochrome poorly, so it was necessary to develop independently an antiserum against labile phytochrome. The antiserum was prepared against proteolytically undegraded phytochrome obtained from etiolated Avena and Pisum seedlings using conventional methods. The antiserum to phytochrome from each of the above mentioned plants was prepared by injecting purified phytochrome into rabbits. The newly produced polyclonal antibodies to phyA from Avena and Pisum were used to characterize phytochrome from etiolated seedlings of Pharbitis nil. The cross reaction was tested by immunobloting. Both kinds of PAbs recognised phyA from Pharbitis nil, however IgG against the labile phytochrome from Pisum gave stronger reaction. The recognized peptide had the molecular weight of about 120-kDa.