Fig. 2.

The unpurified proteins expressed in vitro can be used for serology on immunodot blots or microarrays. (A) Immunodot blots of 112 different vaccinia proteins (encoded by 124 plasmids cloned by colony selection) expressed in vitro. The control reaction lacked plasmid template; empty vector controls generate a positive signal because of the expression of a small 10× His-positive fragment (data not shown). Volumes of 0.3 μl of each reaction were spotted directly onto nitrocellulose in duplicate and probed with either anti-His tag Ab (Upper Left), anti-HA tag Ab (Upper Right) VIG (Lower Left), or VIG plus 10% E. coli lysate (VIG+L; Lower Right). The identity of each pair of spots is given in Table 2, which is published as supporting information on the PNAS web site. Proteins marked with an asterisk in Upper were negative when probed with both anti-His and anti-HA Abs. E. coli lysate unmasks vaccinia proteins recognized by VIG; dots considered positive by visual estimation are indicated. (B) A pilot microarray constituting five vaccinia proteins expressed from DNA with colony selection and probed with VIG, preabsorbed with or without E. coli lysate. Interspot distance, 0.3 mm.