Journal: PLoS ONE

Article Title: Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase

doi: 10.1371/journal.pone.0145872

Figure Lengend Snippet: (A) ELISA for testing the specificities of TA8 and its three variants against the SH3 domains of Btk, Hck, and Lyn. Absorbance (405 nm) values were normalized to the display level of the FLAG-epitope on virions, with error bars corresponding to the standard deviations of triplicate measurements. Casein was the negative target control. (B) Amino acid sequences of the BC and FG loops of TA8, three variants, and wild-type FN3 (WT-FN3). (C) Phage ELISA for examining the specificity of one TA8 variant, 2H7. Microtiter plate wells were coated with equal amounts of SH3 domains (His 6 -tagged), as demonstrated by comparable binding of an anti-His 6 tag antibody to the wells. Virions displaying WT-FN3 served as a negative control for phage-display. The binding of 1F11, a monobody that binds to several SFKs SH3 domains , was used as the internal control to monitor the functions of the immobilized SH3 domains. Error bars are the standard deviations of triplicate measurements.

Article Snippet: After another 12 min incubation at 30°C, each reaction was loaded into three adjacent wells of a non-binding microtiter plate (Corning; New York, NY, # 3915) and scanned by POLARstar OPTIMA plate reader (BMG LABTECH).

Techniques: Enzyme-linked Immunosorbent Assay, FLAG-tag, Control, Variant Assay, Binding Assay, Negative Control