(A) Schematic of the monobody scaffold. The β strands and loops are labeled and the diversified residues are marked as red spheres. The amino acid sequences of the monobody library and monobody clones. In the library designs, ‘X’ denotes a mixture of 30% Tyr, 15% Ser, 10% Gly, 5% Phe, 5% Trp, and 2.5% each of all the other amino acids except for Cys; ‘O’, a mixture of Asn, Asp, His, Ile, Leu, Phe, Tyr, and Val; ‘U’, a mixture of His, Leu, Phe, and Tyr; ‘Z’, a mixture of Ala, Glu, Lys, and Thr (Koide et al. 2012). A hyphen indicates a deletion. (B) Titration curves of Mb(hPRDM14_S4) and Mb(hPRDM14_S14) to human PRDM14 and mouse Prdm14. The error bars are the standard deviation (n = 3). The curves show the best fit of the 1:1 binding model. (C) Binding of Mb(S4) and Mb(S14) expressed on yeast surface to 50 nM of hPRDM14 and its homologues, mouse Prdm14, human PRDM12 and human PRDM6. (D) Competitive binding assay for Mtgr1 and monobodies. Binding of 10 nM Mtgr1 to biotinylated Prdm14 immobilized on streptavidin coated M280 beads in the absence and presence of 500 nM purified monobodies, Mb(S4) and Mb(S14). (E) Co-immunoprecipitation of FLAG–HA tagged Prdm14 expressed in mESC using Mb(S4), Mb(S14), α-FLAG M2 antibody or a negative control antibody (‘IgG’). Antibodies used for Western blotting are indicated with the blots. E, elution; FT, flow through; Mtgr1, myeloid translocation gene related 1.