TaqMan® Protein Assays are an adapted form of PLA™, a proximity ligation assay technology [1,2] that combines antibody-protein binding with detection of the reporter nucleic acid by real-time PCR. We have optimized this technique for use with crude cell and tissue lysates and employ TaqMan® Assays to offer a highly sensitive and specific process for measuring protein expression in small samples.

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

  • Predesigned TaqMan® Protein Assays
    Predesigned TaqMan® Protein Assays were 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.
  • Custom TaqMan® Protein Assay Using Your Antibodies
    The TaqMan® Protein Assays Open Kit includes everything you need, except antibodies to your protein of interest, to build your own TaqMan® Protein Assay.

Choose from Predesigned TaqMan® Protein Assays

TaqMan® Protein Assays help quantitate protein expression using gold standard TaqMan® 5- nuclease assay chemistry, with workflow and sample quantity requirements similar to those for our TaqMan® Gene Expression and MicroRNA Assays. By obtaining protein expression results on the same analytical platform, TaqMan® Assays enable direct correlation of mRNA and/or miRNA expression to protein expression. Choose from 6 ready-to-use TaqMan® Protein Assays for human stem cell pluripotency proteins, as well as the common human markers Cystatin B and ICAM1.

Make a TaqMan® Protein Assay Using Your Antibody

The TaqMan® Protein Assays Open Kit facilitates the construction of TaqMan® Protein Assays. Build your own TaqMan® Protein Assays to targets of interest from biotinylated antibodies quickly and easily. Assays can be built from an immunogen-purified polyclonal antibody preparation that is split into two pools or from matched antibody pairs suitable for ELISA, typically made up of one polyclonal antibody and 1 monoclonal antibody.

TaqMan® Protein Assay Workflow

Workflow Stage Predesigned TaqMan® Protein Assays TaqMan® Protein Assay Using Your Antibody
Assay Preparation NA Build TaqMan® Protein Assays using antibodies specific for the protein of interest. Attach biotinylated antibodies to streptavidin oligonucleotide ”prox-oligos” to make assay probes.
Cell/Tissue Lysis Lyse samples (cells or tissues) using a gentle, 1-step procedure in a buffered non-ionic detergent, with no further sample clean up or purification steps required. Lyse samples (cells) using a gentle, 1-step procedure in a buffered non-ionic detergent, with no further sample clean up or purification steps required.
Binding 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 is brought into proximity when the antibodies on the assay probes bind to two different epitopes on the target protein. The assay probes binds to 2 different epitopes on the target protein via antibody/protein interactions. This brings the oligonucleotides on the assay probe pair into proximity.
Ligation/Inactivation 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 brought into proximity by binding to the target protein. Hybridization of a third oligonucleotide to the oligonucleotide ends of the assay probe pair forms a bridge structure that is ligated using DNA ligase. After the reaction, protease treatment inactivates the ligase.
TaqMan® Fast Real-Time PCR The ligation product serves as a DNA template in the real-time PCR using a TaqMan® Protein Assay. Amplification and detection of the ligation product by real-time PCR.
Data Analysis Analyze TaqMan® Protein Assay data using our free ProteinAssist™ Software package. Analyze TaqMan® Protein Assay data using our free ProteinAssist™ Software package

Life Technologies Publications

Literature Publications

References on Proximity Ligation Assays

  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 USA. 101(22):8420-4. Epub 2004 May 21.