Analysis of Post-translational Modifications
Post-translational modifications (PTM) of proteins can influence the structure, stability, function, interacting partners, and localization of the protein 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.
Phospho-protein 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 a 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 LOD, which mean 10–20 times fewer cells per reaction are needed (Figure 3).
|Figure 1: Phospho-protein detection using TaqMan® Protein Assays.||Figure 2:Format of antibodies utilized for the standard ELISA. ||Figure 3: U2OS cells treated with and without etoposide for 20 hrs in 37° C / 5% CO2 incubator prior to lysis.|