Purification of Phytochrome by Affinity Chromatography on Agarose-Immobilized Cibacron Blue 3GA

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Abstract
The binding of phytochrome to cibacron blue 3GA was utilized to develop a new affinity purification procedure for phytochrome. Brushite-purified phytochrome from rye (Secale cereale cv. Cougar) was bound to agarose-immobilized blue dye in 0.1 M potassium phosphate (pH 7.8), contaminating proteins washed out with 0.5 M KCl, and homogeneous phytochrome eluted with 10 mM FMN. Of the phytochrome applied, 95% bound and 60-65% was eluted, giving a 25-30% yield for the complete 1-day procedure. Affinity-purified rye phytochrome was identical to conventionally purified phytochrome in its behavior on sodium dodecyl sulfate gels, in gel exclusion chromatography, in sedimentation in sucrose density gradients and in its spectral properties.