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  9. Understanding TaqMan® Chemistry-Based Protein Assays

Understanding TaqMan® Chemistry-Based Protein Assays

TaqMan® Protein Assays are an adapted form of PLA™, a proximity ligation assay technology1,2 that combines antibody-protein binding with detection of the reporter nucleic acid by real-time PCR. Applied Biosystems has optimized this technique for use with crude cell and tissue lysates and combined it with TaqMan® chemistry to create a highly sensitive and specific process for measuring protein expression in small samples. Assays have been developed for the detection of OCT3/4, NANOG, SOX2, and LIN28 in human embryonic stem cells, as well as ICAM1 and CSTB to measure relative quantification in human cells.

There are two product choices for TaqMan® Protein Assays-based research:

  • Pre-designed TaqMan® Protein Assays
  • Custom TaqMan® Protein Assay Using Your Antibody


References
1. Fredriksson S. and Landegren U. et al. (2002). Protein detection using proximity-dependent DNA ligation assays, Nat. Biotechnol. 20:473-477.
2. Gullberg M., Landegren U., and Fredriksson S. et al., (2004) Cytokine detection by antibody-based proximity ligation. Proc Natl Acad Sci U S A. Jun 1;101(22):8420-4. Epub 2004 May 21.



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TaqMan® Protein Expression Assays Workflow



Choose from Pre-designed TaqMan® Protein Assays


These assays help quantitate protein expression using gold standard TaqMan® 5' nuclease chemistry with a workflow and sample quantity similar to our TaqMan® Gene Expression and MicroRNA Assays. As the protein expression results are obtained on the same analytical platform, these revolutionary assays enable direct correlation of mRNA and/or miRNA expression to protein expression. Choose from 6 ready-to-use assays for human stem cell pluripotency proteins, as well as the common human markers Cystatin B and ICAM1.

The basic steps are:
  1. Binding of a protein target by paired assay probes
    The assay probes are target-specific antibodies that are conjugated to oligonucleotides through a biotin-streptavidin linkage. Each oligonucleotide in the pair presents a 5′ or 3′ end that are brought into proximity when the assay probes bind to two different epitopes on the target protein.

  2. Ligation of the oligonucleotides by DNA ligase
    The substrate for ligase is a bridge structure formed by hybridization of a third oligonucleotide to the oligonucleotide ends of the assay probe pair. This structure forms preferentially when the assay probes are in proximity to each other.

  3. Amplification and detection of the ligation product by TaqMan® real-time PCR assay
    The ligation product serves as a template in the TaqMan® real-time PCR assay.