Detection and purification of RAP-tagged EGFR by the RAP tag system. (A) Western blot analysis of RAP-tagged EGFR using anti-RAP tag mAb, PMab-2. Total cell lysates (CHO-K1, CHO/EGFR, LN229, and LN229/EGFR) were electrophoresed under reducing conditions using 5%–20% SDS-PAGE gel and transferred to a membrane. The membrane containing the same amount of lysate was immunoblotted using 1 μg/mL PMab-2 (anti-RAP tag), AC-15 (anti-β-actin), or RMab-3 (anti-IDH1) for 30 minutes, and incubated with a peroxidase-conjugated secondary antibody specific for mouse IgG. (B) Flow cytometric analysis of RAP-tagged membrane protein. Two antihuman EGFR mAbs (clones: EMab-51 and AY13) were used in this study. EMab-51 (IgG₁, κ) was established in our laboratory. AY13 (IgG₁, κ) was purchased from BioLegend (San Diego, CA). CHO-K1 cells and CHO/RAP-EGFR were treated with 1 μg/mL PMab-2, EMab-51, or AY13 for 30 minutes at 4°C, followed by 1:1000 diluted Oregon Green 488 goat antimouse IgG (Thermo Fisher Scientific, Inc., Waltham, MA). Fluorescence data were collected using a Cell Analyzer EC800 (Sony Corp., Tokyo, Japan). (C) Sandwich ELISA of RAP-tagged protein. PMab-2 was immobilized at a concentration of 10 μg/mL for 30 minutes. After blocking with 1% bovine serum albumin in 0.05% Tween 20/phosphate-buffered saline (pH 7.4), recombinant PA-EGFR_ec-RAP-MAP was added at a concentration from 3 ng/mL to 10 μg/mL and was incubated overnight. After washing, the plates were incubated with 0.5 μg/mL biotinylated NZ-1 (anti-PA tag), followed by 1:5000 diluted peroxidase-conjugated streptavidin (GE Healthcare Bio-Sciences, Pittsburgh, PA). The enzymatic reaction was performed using 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.). The optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA). Data are means of four replicates ± SEM. (D) Purification of soluble ectodomain fragment of EGFR (EGFR_ec). LN229/EGFR_ec was cultured and 1 L of culture supernatant was harvested. The filtered supernatant was passed through PMab-2-Sepharose (4 mL bed volume), and the same process was repeated three times. The beads were then washed with 80 mL of Tris-buffered saline (TBS; pH 7.5) and eluted with 0.1 mg/mL epitope peptide in a step-wise manner (4 mL × 10). Ten microliters of the 5th of 5 washes in TBS (wash) and 10 peptide-eluted fractions (lanes 1–10) during the column chromatography were subjected to 5%–20% SDS-PAGE under reducing conditions and were stained with Coomassie brilliant blue. Arrow: EGFR_ec. ec, ectodomain; EGFR, epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SS, signal sequence.