HuMab immunoprecipitation of WT and mutant hSOD1 from mammalian cells.
A human derived cell line (293T) was transiently transfected with vectors engineered to express myc-tagged wild type (WT) or mutant hSOD1 (A4V, G85R, or G93A), or with empty vector as a control (neg). (A) Lysate from transfected cells was subjected to SDS-PAGE and immunoblot. Myc tagged proteins were detected with a mouse monoclonal antibody specific for the myc tag followed by goat anti-mouse HRP conjugate and chemiluminescence. An arrow to the right of the blots indicates a band present at the expected size for SOD1 (16 kDa). Lysate from transfected cells was mixed with an irrelevant isotype-matched antibody (B), HuMab 120c (C), and HuMab 11L-80 (D) and incubated at ambient temperature for 2 hrs followed by immunoprecipitation (IP) with protein A sepharose beads. Precipitated material was subjected to SDS-PAGE and immunoblotted with the anti-myc antibody. (E) Additional immunoprecipitations where performed with the remaining HuMabs, and the presence or absence of a band at the appropriate size for SOD1 in anti-myc immunoblots is indicated with a plus or minus.