Ab64 binds to a different FtGroEL epitope than Ab12 and Ab53.

<p>(<b>A</b>) Isotype-specific competition WB. LVS lysate nitrocellulose strips were pretreated with either 800 µg/ml of Ab12 (competitor mAb, when indicated), or were mock-treated, and then 5 µg/ml of Ab12, Ab53 or Ab64 (primary mAb) was incubated with the membrane strips. Anti (a)-m (mouse)-IgG (H+L, binds IgG1, IgG2b and IgG3) or a-m-IgG2a-specific alkaline phosphatase (AP) conjugates (second Ab) were used for development, as indicated. (<b>B</b>) Immunoprecipitation. Twelve µg of the indicated mAb were incubated with 100 µl (0.25 OD₆₀₀) of LVS lysate (+) or just buffer, followed by capture of the antibody: antigen complex with 7.5 µl of Protein G agarose resin. After washing the resin to remove unbound components of the sample, Ab and Ab-bound antigen were visualized by SDS-PAGE on 4–15% polyacrylamide gradient gels under reducing (and boiling) or non-reducing conditions. The positions of prestained molecular weight standards (in kDa) are indicated. The positions of Ab, Ab H and L chains, and GroEL are indicated by arrows. Ft mAbs Ab58 (IgG1, anti-histone-like protein HU form B) and FB11 (IgG2a, anti-LPS) were used as specificity controls.