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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
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.
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.
Workflow Stage | Predesigned TaqMan® Protein Assays | TaqMan® Protein Assay Using Your Antibody |
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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 |
References on Proximity Ligation Assays