Supplementary Figure 10 Biolayer interferometry (BLI) binding profiles of AtzC wildtype SH2 fusion (AtzC9 wtSH2) and AtzC superbinder SH2 fusion (AtzC-SH2) to phosphorylated SH2 binding peptide AtzA fusion (pY-AtzA)

Article: Stimulus-responsive Self-Assembly of Protein-Based Fractals by Computational Design

Pre-print: bioRxiv 274183; doi: https://doi.org/10.1101/274183

Figure: S10. Biolayer interferometry (BLI) binding profiles of AtzC wildtype SH2 fusion (AtzC9 wtSH2) and AtzC superbinder SH2 fusion (AtzC-SH2) to phosphorylated SH2 binding peptide AtzA fusion (pY-AtzA). (A) Binding profile of AtzC-wtSH2 to pY-AtzA. PY-AtzA was loaded onto the biosensor via a streptavidin-biotin interaction. AtzC-wtSH2 was flowed into the sample. KD = 41.79 ± 0.32 nM. (B) Binding profile of AtzCM1 (superbinder) to pY-AtzA. PY AtzA was loaded onto the biosensor via a streptavidin-biotin interaction. AtzC-SH2 was flowed into the sample. KD = 7.67 ± 0.52 nM.

Comment. KD was calculated using ForteBio BLItz software. KD statistical analysis provided in datafile.

(SI 2.8) Bio-layer interferometry (BLI) – AtzAM1 was phosphorylated using the conditions described below. pY-AtzAM1 was then biotinylated at 10mM Sulfo-NHS-Biotin (APExBIO) for 30min at 25°C. Excess biotin was buffer exchanged with a PD-10 desalting column (GE Healthcare) equilibrated with HNG. Biotinylated pY-AtzAM1 was loaded onto streptavidin (SA) coated biosensors (ForteBio) and used for BLI. AtzCM1 was flowed in from 4nM to 4μM. BLI experiments were performed using the BLItz System (ForteBio).
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Note: This work was part of the Rutgers Biomod 2016 Project (M. Liu, A. Permaul, O. Dineen, M. Khalid, M. Shea, G.L. Bilker). See reference below.