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
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.
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:
- 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.
- 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.
- 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.