Fig. 1

In-solution SH2 domain binding assay using proximity ligation and real-time PCR. a , Schematic Illustration of SH2-PLA. A pair of PLA probes is used to detect the interaction of tyrosine phosphorylated EGFR and a GST-SH2 domain. The 3′ SH2-PLA probe consists of an anti-EGFR antibody conjugated with the 3′ proximity oligonucleotide (3′ Prox-Oligo). The 5′ SH2-PLA probes consists of an anti-GST antibody conjugated with the 5′ Prox-Oligo and a GST-SH2 domain. When the GST-SH2 domain binds to tyrosine phosphorylation sites of EGFR, the 5′ and 3′ PLA probes are brought in close proximity, allowing ligation of the two Prox-Oligos which is detectable by real-time PCR. b , Experimental workflow of SH2-PLA Method 1. Lysates are prepared with or without EGF stimulation. Biotinylated anti-GST and anti-EGFR antibodies are conjugated with the 5′ and 3′ Prox-Oligos, respectively, and stored at −20 °C. The 5′ SH2-PLA probe is mixed with purified GST-SH2, and the 3′ SH2-PLA probe is mixed with cell lysates allowing the antibodies to bind their respective epitopes. Subsequently, the 5′ and 3′ PLA probe solutions are combined to induce interaction between the SH2 and pEGFR. Then, the amount of the complex is quantified by proximity ligation and real-time PCR. An alternative method is also possible (Additional file 1: Figure S2). Estimated assay runtime including sample-handling steps for each procedure is noted on the right