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. 2016 May 17;17(10):900-3.
doi: 10.1002/cbic.201600068. Epub 2016 Apr 1.

Multiplexed Imaging of Protein Phosphorylation on Membranes Based on Ti(IV) Functionalized Nanopolymers

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Free PMC article

Multiplexed Imaging of Protein Phosphorylation on Membranes Based on Ti(IV) Functionalized Nanopolymers

Anton Iliuk et al. Chembiochem. .
Free PMC article

Abstract

Accurate protein phosphorylation analysis reveals dynamic cellular signaling events not evident from protein expression levels. The most dominant biochemical assay, western blotting, suffers from the inadequate availability and poor quality of phospho-specific antibodies for phosphorylated proteins. Furthermore, multiplexed assays based on antibodies are limited by steric interference between the antibodies. Here we introduce a multifunctionalized nanopolymer for the universal detection of phosphoproteins that, in combination with regular antibodies, allows multiplexed imaging and accurate determination of protein phosphorylation on membranes.

Keywords: antibodies; dendrimers; membranes; multiplexed analysis; phosphoproteins.

Figures

Figure 1
Figure 1
A) Simplified procedure schematic for pIMAGO-Fluor-based detection of phosphoproteins by western blot. B) Multiplexing proposal of pIMAGO-Fluor with an antibody, demonstrating apparent size difference between pIMAGO and an antibody.
Figure 2
Figure 2
A) pIMAGO-Fluor based detection of six-protein mixture separated by SDS-PAGE in amounts ranging from 400 to 0.39 ng for each protein. The two phosphorylated proteins are denoted by “P”. B) Graphic representation of detection linearity and quantitation for β-casein in (A).
Figure 3
Figure 3
Multiplexed detection of Cdk1 + Rb (A and B) and JNK1 + cJun (C and D) in in vitro kinase assays. The assay mixtures (with and without ATP) were separated by SDS-PAGE and detected in the 700 channel (red) by using pIMAGO-Fluor (A and C) or phospho-site-specific antibodies (B and D). Each membrane was also subsequently incubated with general anti-Rb or anti-cJun antibodies and detected in the 800 channel (green), and the signals were overlaid (yellow).
Figure 4
Figure 4
Multiplexed detection of Syk+Cdb3 in an vitro kinase assay. The assay mixtures (with and without ATP) were separated by SDS-PAGE and detected in the 700 channel (red) by using A) anti-pTyr antibody or B) pIMAGO-Fluor. Each membrane was also subsequently incubated with general anti-Cdb3 antibody and detected in the 800 channel (green), and the signals were overlaid (yellow). Numbers above the signal bands show the ratio differences compared to the control in (C). C) As a control for signal normalization, the kinase assay was also detected by anti-Cdb3 antibody alone. D) Quantitative representation of the ratio differences between pIMAGO-Fluor and anti-pTyr effects on subsequent anti-Cdb3 signal. The averages and error bars of standard deviations are based on seven different experiments (ns = differences were not statistically significant, *= differences were statistically significant at t-test p < 0.05).
Figure 5
Figure 5
Multiplexed detection of protein A-tagged Rad9 (A) and Rad17 (B) affinity purified samples before and after treatment with DNA damage agent MMS. pIMAGO-Fluor was used to detect phosphorylation (red) and antiprotein A antibody for total protein amount (green). Numbers above the signal bands show the ratio differences compared to the untreated control after normalization with protein.

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