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Preparative SDS-PAGE Electrophoresis of a Recombinant Epstein-Barr Virus Encoded Protein and Its Application in Serodiagnostic Test Systems, Rev C

buffer, 8 M urea) was dialyzed extensively against Tris-HCl (50 mM, pH 8.0). The white precipitate was collected and boiled in 1 ml sample buffer for 5 min. The sample was loaded onto a 12% cylindrical SDS-PAGE gel and electrophoresed as indicated in Table 1. Collection of 2 ml fractions was started when the Bromophenol Blue band passed the gel matrix.

Following electrophoresis, 200 l aliquots of every fifth eluted fraction were TCA-precipitated and analyzed by Coomassie Blue-stained SDS-PAGE and immunoblotting for the presence of p23-DHFR (Figure 2). As confirmed by high-sensitivity immunoblot analysis, the protein preparation was purified to near homogeneity in the course of the electroelution procedure. Only a single protein band, corresponding to the 49 kD recombinant p23-DHFR, was visible in lanes 79 (Figure 2B).

Estimation of protein concentration demonstrated that the p23-DHFR-positive fractions contained approximately 10% of total applied protein. As urea was removed during the electrophoresis procedure, only renatured recombinant protein could be eluted in a soluble form. The decrease in yield is probably due to precipitation of insoluble protein within the PAGE column.

Immunoblot Analysis
To evaluate the diagnostic potential of the EBV-encoded recombinant protein in a more systematic manner, immunoblot and ELISA tests were carried out to screen a representative panel of EBV-positive and -negative human sera for anti-p23 IgM- and IgG-specific antibodies.

For immunoblot analysis, the purified p23-DHFR protein preparation (Figure 2B, lane 9) was electrophoresed and transferred to a 0.45 m nitrocellulose membrane via blotting (Trans-Blot SD semi-dry transfer cell). Following
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