FIG. 3.

Identification of MALP-404 in TX-114 phase-fractionated proteins from M. fermentans PG18. (A) Total proteins from the TX-114 phase were separated by SDS-PAGE, transferred to nitrocellulose, and immunostained with preimmune mouse serum (lane 2), hyperimmune PAb generated to the MALP-2 synthetic peptide (lane 3) or to a C-terminal synthetic peptide within the MALP-404 ORF (lane 4), hyperimmune PAb to gel-excised 41-kDa protein (lane 5), MAb F208C2B1 to the MALP-2 synthetic peptide (lane 6), or MAb 4444H7.A to a previously described (59) 41-kDa protein (lane 7). As a control to indicate sizes of other proteins in this fraction, a blot strip was also stained with a combination of MAbs to amphiphilic proteins P78, P76, P61, and P38 (lane 1) described previously (59) or in this report (protein P76). (B) Immunoprecipitation of MALP-404 and localization of multiple epitopes on the product. The abundant 41-kDa amphiphilic protein recognized by MAb 4444H7.A was immunoprecipitated with this MAb from TX-114 phase proteins, as described in Materials and Methods. Immunoprecipitated protein (along with precipitating MAb) was separated by SDS-PAGE, transferred to nitrocellulose, and immunostained with a series of Abs, including MAb 4444H7.A used for immunoprecipitation (lane 1), PAb to gel-excised P41 (lane 2), MAb F208C2B1 to the MALP-2 synthetic peptide (lane 3), or PAb to the C-terminal synthetic peptide of the MALP-404 ORF product (lane 4). A negative control, showing the absence of staining by preimmune mouse antisera is also shown (lane 5). The positions of the immunoprecipitating immunoglobulin heavy (H) and light (L) chains (which are lightly stained by the secondary Ab reagent) are indicated on the left.