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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).