( Poinceau Red staining membranes ...)Preparative SDS-PAGE Electrophoresis of a Recombinant Epstein-Barr Virus Encoded Protein and Its Application in Serodiagnostic Test Systems, Rev C

Poinceau Red staining, membranes were washed twice with phosphate buffered saline (PBS, pH 7.2), and nonspecific binding of antibodies to the membrane was blocked by incubation with 5% nonfat milk in PBS for at least 1 hr at room temperature. After being washed three times with PBS/Tween (PBS containing 0.05% [v/v] Tween 20), the membranes were subsequently incubated with diluted human sera (1:100 in 5% nonfat milk/PBS) for 1 hr. Following serum incubation, blots were washed three times with PBS/Tween and incubated with horseradish peroxidase-conjugated goat anti-human IgG antibody. After a 2 hr incubation at room temperature, blots were washed three times with PBS/Tween, and color was developed with HRP color development reagent DAB.

As illustrated in Figure 3, p23-DHFR was highly antigenic with both NPC (n2267, n243) and other EBV-positive sera (1731 to 1624), and showed no reactivity with EBV-negative sera (1626, 1197). Again, the purity of the protein preparation was demonstrated with these individual sera, as not even marginal reactivities were observed to any proteins different from the 49 kD recombinant antigen.

Conclusions
This report describes the purification of a recombinant antigen that was expressed in E. coli cells at such a high level that it constituted up to at least 50% of total cellular protein. After sonication, most of the recombinant protein was located in the insoluble fraction. Initial purification of the recombinant protein was performed by fractioned solubilization in buffers containing rising concentrations of urea. Optimization of this technique can overcome, in some cases, the application of tedious and yield-reducing stan
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