Proteins coimmunoprecipitated and coeluted with TAPP2. (A) Protein extract from 160 × 106 BJAB cells overexpressing untagged TAPP2 were used in scaled-up TAPP2 immunoprecipitations, followed by elution with 10 μg/mL TAPP2 epitope peptide. Where indicated, cells were stimulated for 10 minutes with 10 μg/mL anti-BCR before protein isolation. Bead-bound and eluted fractions were separated on 7% SDS-PAGE gels and stained with SilverSnap mass spectrometry–compatible protein stain. The white arrow indicates a 47-kDa band in TAPP2 eluates (obscured by the heavy chain of the IP antibody in the bead fractions). This band was excised and identified as TAPP2 by mass spectrometry. indicates a frequently observed high-molecular-weight band present only in TAPP2 IP lanes and eluting with TAPP2 epitope peptide. (B) The band indicated by the was excised, destained, processed with trypsin, and identified by mass spectrometry peptide fingerprinting as the cytoskeletal protein utrophin. The figure shows the amino acid sequence of utrophin, indicating the 27 unique tryptic peptides identified, spanning the protein sequence.
