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Fig. 3 | BMC Biotechnology

Fig. 3

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

Fig. 3

Estimation of EGFR phosphotyrosines at the limit of detection. To define an absolute lower limit of detection (LOD) for SH2-PLA, the total amount of EGFR phosphotyrosines in sample cell lysate was estimated using a phosphotyrosine standard sample and quantitative dot blotting analyses. a, Recombinant c-Abl protein, the pTyr-standard sample, was treated with tyrosine specific phosphatases PTP1B and TC-PTP. The amount of hydrolyzed phosphotyrosine was quantified by malachite green phosphatase assay (∆Abl PO4 3−). Left panel shows anti-Abl and anti-phosphotyrosine blots for phosphatase-treated (Abl PTP+) and -untreated (Abl PTP-) samples. After the PTP treatment, the level of c-Abl tyrosine phosphorylation was greatly reduced but weak phosphorylation was still detectable with longer exposure time. The right panel shows a plot of the phosphate standard used for the quantification. Red circle, untreated c-Abl; blue circle, PTP-treated c-Abl; yellow circles, the kit supplied phosphate standard. From this analysis, ∆Abl PO4 3 was estimated to be 5.7 pmol per μg of the c-Abl protein. b , Quantitative dot blotting. The total pTyr in the EGF-stimulated Cos1 cell lysate was estimated from a pTyr standard curve generated from anti-phosphotyrosine dot blotting. Upper panel shows raw anti-Abl and anti-pTyr blots. Serially diluted c-Abl pTyr standard (left to right 3.1–0.02 ng per spot) and 0.01 μg EGF-stimulated Cos1 samples were spotted on nitrocellulose membrane (performed in triplicate). The middle panel shows the resulting pTyr standard plot with the quantified signal intensities. The pTyr amount in the EGF-stimulated Cos1 lysate was estimated to be 0.08 pmol per μg lysate. Subsequently, an anti-pTyr Western analysis for A431 and Cos1 samples was performed, relative intensities of the EGFR bands were calculated, and the amount of EGFR pTyr in the EGF-stimulated A431 sample was estimated to be 0.122 pmol/μg. Thus 2 ng of EGF-stimulated A431 sample, which is the lower limit for SH2-PLA detection, would contain 0.243 femtomole EGFR pTyr. See Methods and Additional file 1: Figure S4 for more information

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