Analysis of Post-Translational Modifications

Post-translational modifications (PTM) of proteins can influence their structure, stability, function, interacting partners, and localization within the cell. There are numerous methods available to measure post-translational modifications, including western blots, ELISAs, and mass spectrometry. Because of the specificity, sensitivity, and small sample requirement; TaqMan® Protein Assays are an appealing alternative to more cumbersome and less quantitative methods. TaqMan® Protein Assays offer highly sensitive target phosphoprotein expression analysis with just a few thousand cells per well.

Phosphoprotein detection using TaqMan® Protein Assays requires two different proximity probes, each containing a different antibody. One antibody is targeted to the phosphorylation site of interest, and the second is targeted to another epitope on the protein (Figure 1).

Benchmarking versus a standard colorimetric detection ELISA with colorimetric detection (Figure 2) was performed in the cell line U2OS to demonstrate the performance of the TaqMan® Protein Assay for characterizing endogeneous expression of phosphorylation sites in cells.

This benchmarking demonstrated that TaqMan® Protein Assays are about 10–20 times more sensitive than ELISA in terms of limit of detection (LOD). This means that 10–20-fold fewer cells per reaction are needed for analysis with TaqMan® Protein Assays compared to ELISA (see figures).