Analyzing Proteins with PCR
TaqMan® Protein Expression Assays from Applied Biosystems, part of Life Technologies, “open up new territory for real-time PCR,” facilitating quantification and analysis of protein expression as well as analysis of protein-protein interactions and pathway responses resulting from post-translational phosphorylation events, contends David Ruff, Ph.D., principal scientist at Life Technologies.
At the heart of this homogeneous assay system are two antibodies, one of which contains a bound 3´ open end of an oligonucleotide and the other the 5´ open end of a different oligonucleotide. Each antibody recognizes a distinct epitope on the protein of interest. When both antibodies are bound to the target, their conjugated oligos are brought into close proximity; ligation of the oligos is then directed with a connector oligo sequence creating an amplicon template for primer and probe hybridization for real-time PCR amplification.
The antibody pair sets can be monoclonal or polyclonal antibodies; each antibody is labeled with biotin. They are conjugated to streptavidin-linked oligonucleotides. The entire workflow, encompassing preparation of the whole cell lysate, binding of the oligo-antibody probes to the target proteins in the wells of a microplate, ligation and inactivation of ligase, and PCR takes about 3.5 hours, according to Dr. Ruff.
The assay “requires very little material, about three orders of magnitude less than for a Western blot.” Whereas the limit of detection of a Western blot is typically 30,000–10,000 cells/lane, it is typically 10–35 cells/well for the protein assay. Using the resulting CT values, the research “can correlate protein to mRNA levels on one platform.”
Dr. Ruff presented an example of how the assay could be used to detect and quantify changes in expression of protein markers of stem cell pluripotency and differentiation. Another application he described used the Protein Expression Assay to measure changes following siRNA knockdown of a target mRNA. He provided data showing a good correlation between the results of an in situ hybridization experiment and the corresponding protein expression assay results.
Life Technologies’ current product line includes six assays for stem cell regulatory proteins. In development and expected to launch in mid-2010 is the TaqMan Protein Expression Assay Open Kit, for which customers will supply the biotin-labeled antibodies for their protein target of interest, and the company will provide the streptavidin-labeled oligos and Universal assay reagents for ligation and qPCR.