Fig. 6: One-step purification of Azo-tagged POIs in parallel using the 96-well format.

A Isolation of the β-lactamase AmpC carrying the C-terminal GG-Pap sequence from the total cell extract of NEBExpress(lowRF1) using 10 wells of a 96-well receiver plate filled with 200 µl α-CD affinity resin each. AmpC was purified as essentially homogeneous protein without significant well-to-well difference. AmpC without an Azo-tag or untransformed NEBExpress(lowRF1) cells served as negative controls. B Colorimetric assay to measure the AmpC enzyme activity in aliquots of the eluate from (A) after mixing with CENTA substrate in a 96-well microtest plate. C Affinity purification of the anti-CEA scFv antibody fragment T84.66 carrying the C-terminal GG-Pap sequence from the periplasmic extract of NEBExpress(lowRF1), followed by ELISA to assess antigen-binding activity in 10 separately eluted aliquots. scFv bound to a CEA-coated microtiter plate was detected via an antibody-HRP conjugate directed against the Strep-tag II using ABTS substrate. Untransformed NEBExpress(lowRF1) cells served as negative controls (3 wells). Data points in box plots shown in (B) and (C) each correspond to a single measurement per well; median (center line), upper/lower quartiles (box limits) and min/max values (whiskers) are indicated. D Light-controlled affinity purification of trastuzumab from cell culture medium using an adapter protein, as schematically illustrated to the right. Trastuzumab and the Azo-tagged protein G C2 fragment (Azo-ProtG) were mixed in DMEM, 5 % (v/v) FCS (input). The antibody was specifically isolated by UV light-controlled elution while all serum proteins, in particular albumin (∼66 kDa), were efficiently removed.