Figure 5.

Isolation of sV23 disulfide-linked complexes by Ni-affinity chromatography.

(A) Enriched eggshells from 900 stage 10 egg chambers from VM26Ab^QJ42/VM26Ab^QJ42 females carrying two copies of the sV23-His transgene were resuspended in denaturing buffer (I-input) and applied to a packed 100 ul Ni-IDA affinity column. Six 80 ul wash fractions (w) were used to collect unbound proteins; following the addition of 250 mM imidizole, six 80 ul elution fractions (e) were collected; 4 column volumes of denaturing buffer containing 2% SDS pre-heated to 95° [(s) – 80ul/fraction] were used to collect residual proteins. After the addition of Laemmli sample buffer and β-ME, proteins from a small portion (0.6%) of selected fractions were separated by SDS-PAGE. The upper panel shows a blot incubated simultaneously with sV23 and sV17 antisera. The lower panel shows a blot of the same column fractions incubated with a DEC-1 antiserum (Cfc106). The positions of sV23, sV17, and CRM-80 (cross reacts with the sV23 antiserum) are shown to the left. In the lower panel the positions of the DEC-1 fc106 derivatives, s80 and s60 are indicated. (B) Silver stained gel of 12.5% of each fraction shown in (A) [Note: the band observed in the w5 and e2 fractions was not reproducible]. (C) As in (A) except enriched eggshells were prepared from 200 wild type stage 10 egg chambers and 3% of each fraction was analyzed. (D) As in (A) except 200 stage 10 egg chambers were used to prepare the enriched eggshells and the enriched eggshells were resuspended in denaturing buffer containing 5mM βME. The upper panel shows a blot incubated with the sV23 antiserum; the lower panel shows the reactive region of a parallel blot incubated with the sV17 antiserum. (E) Western blot of SDS soluble proteins from wild type (wt) and Vml^EPgy2 (vml) stage 10 egg chambers incubated with the sV23 antiserum. (F) Western blot of sample processed as in (A) above except the enriched eggshells were from 250 stage 10 egg chambers derived from homozygous Vm26Ab^QJ42 females carrying two copies of a sV23-SSC-His transgene.