Fig. 4.

SEC using the NTR1 mutant D03 (Sarkar et al., 2008) and the selected DARPin P28. Solid line: elution profile of a mixture of D03 (3 nmol) and the DARPin P28 (10 nmol); dashed line: elution profile of the receptor alone; dotted line: elution profile of the DARPin P28 alone. Inset: western blot analysis of TCA-precipitated fractions of the corresponding peaks. Lane 1 corresponds to peak (1) of the mixture, lane 2 to the peak labeled (2) of the GPCR and lane 3 to the DARPin run alone, peak (3). The proteins were separated by SDS–PAGE, transferred to a PVDF membrane and detected by a mixture of an anti-rNTR1 (R-20) antibody (Santa Cruz Biotechnology, #sc-7598) and an anti-RGSH₄ antibody (for DARPin detection). It should be noted that the DARPin carries a 65-aa C-terminal 5-fold Myc-tag which is highly charged and presumably disordered. We have previously observed that this significantly increases the hydrodynamic radius of these already elongated molecules.