Understanding TaqMan® Chemistry–Based Protein Assays
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|
- Guide: Designing TaqMan® Protein Assays Using Your Antibodies
- Brochure: TaqMan® Protein Assays
- Product Overview: TaqMan® Protein Assays
- Application Note: Detection and Relative Quantification of Protein Markers in Embryonic Stem Cells
- Application Note: Analysis of MicroRNA and Protein Expression on a Single Platform Using the ViiA™ 7 Real-Time PCR System
Life Technologies Publications
- Research Note: Analysis of microRNA and Protein Expression on a Single Platform
- Poster: A Novel Quantitative PCR Based Protein Detection Method
- Poster: Novel Protein Expression Assays using qPCR for the Detection and Relative Quantification of Protein Markers in Stem Cells
- Poster: An Integrated Quantitative PCR Approach for Monitoring Gene and Protein Expression in Human Pluripotent and Differentiated Cells
- Ruff D et al. (Epub ahead of print, 2011). Applications of Quantitative PCR Protein Assays During Reprogramming. Stem Cells Dev. 2011 Apr 8.
- Gillis AJ et al. (Epub ahead of print, 2011). Expression and interdependencies of pluripotency factors LIN28, OCT3/4, NANOG and SOX2 in human testicular germ cells and tumours of the testis. Int J Androl. 2011 Jun 2. doi: 10.1111/j.1365-2605.2011.01148.x.
References on Proximity Ligation Assays
- Fredriksson S. and Landegren U. et al. (2002). Protein detection using proximity-dependent DNA ligation assays. Nat. Biotechnol. 20:473-477.
- 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.