FIG. 6.

P protein dimer is stable over a wide range pH and under high-salt conditions. (A) Detections of P protein dimer (D) and monomer (M) at different pHs. Recombinant VA387 P proteins were treated with different pH solutions and then were cross-linked by 30 μM BS3. The treated samples were separated on SDS-PAGE gels. (Left) A Brilliant Blue-stained SDS-PAGE gel. (Right) Western analysis of the P protein after treatments at different pHs and BS3 using antibody against VA387 capsid protein. The different pH solutions are indicated for each lane. Stand, prestained protein standard (broad range; Bio-Rad); control, untreated and un-cross-linked P protein. (B) Binding assay of the pH-treated P protein to A-type saliva. (C) Immunodetection of P protein dimers after denaturation and/or renaturation. P proteins were denatured (de) by 8 M urea (U), 6 M guanidine (G), or extreme high or low pHs. Before (de) and after (re) removal of the denaturation factors, samples were cross-linked by 30 μM BS3. The treated samples were separated on an SDS-PAGE gel. Control, untreated and un-cross-linked P protein; U-de, 8 M urea-treated P protein; U-re, renatured P protein after removal of the 8 M urea; G-re, renatured P protein after removal of the 6 M guanidine; pH 1.5-de, protein denatured by pH 1.5 solution; pH 1.5-re, renatured protein after neutralization from pH 1.5; pH 12.5-de, protein treated by pH 12.5 solution; pH 12.5-re, renatured protein after neutralization from pH 12.5. The BS3 concentration was 30 μM. Each lane contained ∼800 ng of P protein.