FIGURE 2.

Expression and purification of monomeric and dimeric forms of IgA2 b12. A, Nonreducing SDS-PAGE analysis of monomeric, dimeric, and polymeric forms of IgA₂ b12, as compared with IgG1 b12. Ab preparations were size-fractionated by nonreducing SDS-PAGE and stained with Coomassie blue. Monomeric IgA₂ b12 migrated with a molecular mass of ~150 kDa (filled arrowhead), whereas higher molecular mass forms (>220 kDa; open arrowheads) were present in the dimeric and polymeric IgA b12 preparations. A protein band of ~50 kDa was present in all IgA b12 preparation (*), and likely corresponds to κ L chain dimers that dissociate from intact Ig (52). IgG1 b12 migrated with a molecular mass of ~160 kDa. B, Separation of dimeric and monomeric forms of IgA₂ b12 by FPLC gel filtration. As described in Materials and Methods, Abs were eluted from by isocratic elution on HiPrep 16/60 Sephacryl S-300 High Resolution column at a flow rate of 0.5 ml/min. Peaks containing dimeric IgA₂ b12 (50.02 peak) and monomeric IgA₂ b12 (61.52 peak) were determined by ELISA and SDS-PAGE. The following molecular mass standards were used to calibrate the column: dextran 2000 kDa, ferritan 440 kDa, catalase 220 kDa, and RNase A 10 kDa. C, Anti-J chain Western blot analysis. Monomeric, dimeric, and polymeric forms of IgA₂ b12, as well as IgG1 b12, were subjected to nonreducing SDS-PAGE, transferred to nitrocellulose, and probed with an anti-human J chain Ab. The minor band observed in the IgG1 b12 lane is probably due to spill over from the adjacent pIgA₂ b12 sample.