MATERIALS AND METHODS

The procedure of Ryan et al. (1985a) was used to purify the arylphorin. The yield of arylphorin during purification was critical because of the tiny amount of protein that the incubated body secretes in the first place. The procedure of Ryan et al. (1985a) has one significant downfall in this respect: it involves many steps which result in a loss of yield. The procedure was modified slightly because of this.

The arylphorin (which came from incubated fat body) was mixed with a cold buffer. After centrifugation, the suspension was frozen and thawed; this denatured the contaminants which were then removed. The supernate was concentrated and then subjected to lectin affinity chromatography.

Most contaminating protein eluted with polyacrylamide slab gel electrophoresis with a buffer alone. Density scanning was used and showed that one fraction was highly contaminated. Fractions were pooled together to obtain about 98% arylphorin. The yield was about 30% of the initial total protein, which is a higher yield than in the original, unchanged procedure. Specific activity was determined after purification and then the precipitate was solubilized by heating prior to liquid scintillation counting.


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SCOTT SMORRA, NAT JOHNSON, CRISTINE BERRY, MARINELA RODRIGUEZ, MARY OLIVER, TARA DAVIS, ELYSE EARNEST
GROUP 12 DECEMBER 1, 1996