Fig. 1. New add mutants identified by colony-blot immunoassay screening. Representative immunoassay performed on each gene deletion strain as described in Materials and Methods. Rows (a-l) show, in duplicate from left to right, colonies of deletion mutants #01-06 (a), 07-12 (b), 13-18 (c), 19-24 (d), 25-30 (e), 31-36 (f), 37-42 (g), 43-48 (h), 49-54 (i), 55-60 (j), 62-67 (k), 68-73 (l). Row (s) shows duplicates of the M, Z and O parent strain standard controls from left to right, expressing wild-type A1PiM or mutant A1PiZ, or the pYES2.0 vector with no gene insertion. Those deletion mutants exhibiting a darker colony spot than that of the BY4742 WT parent expressing A1PiZ were selected as putative ERAD mutants and re-assayed to eliminate false-positive results. Only deletion mutants 06, 37, 39, 66, 67, 68 and 72 displayed a consistent signal darker than the BY4742 WT parent (Z) in subsequent colony-blot immunoassays. For example, deletion mutants 59 and 60 were selected from this assay but were eliminated by at least two negative results in future assays.

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