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Fig. 2 | BMC Biotechnology

Fig. 2

From: SH2-PLA: a sensitive in-solution approach for quantification of modular domain binding by proximity ligation and real-time PCR

Fig. 2

Validation of the SH2-PLA assay. a , Representative PCR amplification plot for SH2-PLA experiments. Increased binding between SH2 and pEGFR upon EGF stimulation is expressed as a reduced threshold cycle value (Ct). Here, ∆Ct is defined as [Ctcontrol – CtEGF stimulated]. b, Specificity of SH2-PLA. SH2-PLA (top panel) and far-Western (middle panel) results for EGF-stimulated and control A431 cell samples are shown. Results for GST control probe are shown in lanes 1–4; Grb2 SH2 probe in lanes 5–8; and PLCγ1 tandem SH2 probe in lanes 9–12. Lanes 3, 4, 7, 8, 11, and 12 show the assay result in the presence of pY1068 blocking peptide, which contains the Grb2 SH2 consensus binding site of EGFR. c , SH2-PLA assay performance. The SH2-PLA assay was performed three times using a two fold dilution series of EGF-stimulated and control A431 cell lysates. Average Ct values, normalized to non protein control (NPC), are shown in the upper panel. The intra-assay variation for Ct values was 0.07-2.36 (mean 0.60) and the inter-assay %CV was 0.32-3.08 (mean 1.28). Since EGF-stimulated samples always showed a greater signal (lower Ct) than the unstimulated control throughout the dilution series, the range of assay detection is estimated to be at least 1.1–1100 μg/ml of lysate concentration, and the lower limit of detection is approximately 2 ng of protein per assay. The lower panel shows the approximately linear region of the mean Ct plot against log input lysate concentrations, and the ∆Ct (unstimulated – stimulated) of about three cycles. The log2 fold change between EGF-stimulated and control samples was estimated to be 6.0 - 6.4 using the ProteinAssist software tool (Additional file 1: Figure S3). d, Adoption of other phosphotyrosine recognizing domains. The SH2-PLA methodology was applied to protein tyrosine phosphatase (PTP) and phosphotyrosine binding (PTB) domains. ShcA PTB domain and the substrate-trapping mutant of PTP1B PTP domain displayed activity comparable to Grb2 SH2 (lanes 1–4 and 7–8). Signal was undetectable for the wild type (wt) PTP1B PTP domain, likely due to the intrinsic phosphatase activity (lanes 5–6)

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