Table 1.

Summary of phage-display selection methods. Six different phage-display screens were carried out using biotinylated EPAC1ΔDEP as a target (screens S01–S06). Pre-panning deselection and off-rate deselection was performed in pans 2 and/or 3 as a process of negative selection. Here, homologous proteins, (EPAC1ΔDEP with no ligand) were premixed with the phage library, either as immobilized proteins and/or as proteins in solution. Off- rate deselection involves allowing the Affimer-expressing phage to bind to the target, washing away unbound phage and incubating the phage bound to immobilized target overnight in buffer before eluting the following morning; this drive selection towards Affimer proteins with the slowest off-rates. Enrichment refers to the number of captured phage with affinity for biotinylated EPAC1ΔDEP, as indicated by number of positive, phage-transformed bacteria following the panning step compared to a negative selection well.

Screen	Page Display Method			Phage-Display Enrichment
	Target	Pre-Panning Deselection	Off-Rate Deselection
S01	EPAC1ΔDEP	None	In pans 2 and 3	9 × over neutravidin
S02	EPAC1ΔDEP plus 007	EPAC1ΔDEP in Pans 2 and 3	In pans 2 and 3	1.2 × over EPAC1ΔDEP
S03	EPAC1ΔDEP plus CE3F4	EPAC1ΔDEP in Pans 2 and 3	In pans 2 and 3	1.2 × over EPAC1ΔDEP
S04	EPAC1ΔDEP	None	In pan 3	~2 × over neutravidin
S05	EPAC1ΔDEP plus 007	EPAC1ΔDEP in Pans 2 and 3	Not performed	2.5 × over EPAC1ΔDEP
S06	EPAC1ΔDEP plus CE3F4	EPAC1ΔDEP in Pans 2 and 3	Not performed	1.4 × over EPAC1ΔDEP