TaqMan® Protein Assay Support—Getting Started
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An ELISA is used with a standard curve for absolute quantification of an analyte. It often requires numerous wash and incubation steps. TaqMan® Protein Expression Assays are fast, homogeneous assays used for relative quantification. The relative amount of a target protein in a test sample is determined in relation to a reference sample
The largest protein we have tested was ~200 kDa. The distances spanned by the oligo linkers are very large and would suggest that even much larger proteins could be used.
The oligo linker spans a distance of ~50 nm. This is a very large distance compared to a typical folded protein (e.g., 150 kDa antibody is ~ 8 nm in diameter).
This is dependent on the particular assay. The typical dynamic range is 2–4 logs. Some assays will perform better than ELISAs, but others may not.
In some cases you may have to biotinylate the antibody yourself using a commercial kit. Please follow the guidelines in the Probe Development Protocol (Appendix B: Select and Prepare Nonbiotinylated Antibodies). In particular, you will need to pay attention to whether the antibody is resuspended with a carrier protein or an amine-based buffer. Recommendations for the appropriate biotinylation kits are provided in this manual.
Yes, you will have to use a biotinylated antibody for the ligation to occur successfully with the streptavidin proximal oligos and linker. If you cannot find a biotinylated antibody, you can purchase the nonbiotinylated antibody and a biotinylation kit, or utilize our custom services to have it done for you.
Every antibody is different, so there is no predictor for whether a certain monoclonal or polyclonal antibody would work better (provided they both meet the antibody requirements). In terms of setup, using a polyclonal antibody can be easier as you just have to split the pool of antibody between the 5’ and 3’ proximal oligos.
Check out our blog post on “What You Need To Start Using TaqMan® Protein Assays” for a complete list to get started.
Any real-time PCR instrument that can perform fast cycling and detect FAM™ dye can be used with the TaqMan® Protein Assays. However, the data are directly compatible with the ProteinAssist™ Software for downstream analysis only if they are from an Applied Biosystems® real-time instrument. The following instruments have been validated with the software: 7500F (*.eds file, v2.0.2 and later), 7500F (*.csv, v1.4), 7900HT Fast (v2.3), StepOnePlus™ (v2.1 and later), ViiA™7 (v1.0 and later) systems. If you have a different system, please consult firstname.lastname@example.org for more information.
NPC stands for no-protein control. This is a sample (lysate or just buffer) in which your protein of interest is not present. This is a required sample type for the Relative Quantitation analysis in ProteinAssist™ Software.
The first time you use a new assay probe pair, you will want to determine the linear range of the assay. To do so, create a 2- or 3-fold dilution series of your input. For cell lysates, we suggest starting with 500 cells or 50 ng of total protein per well. For tissue lysates, you can use as much as 1,000 ng of total protein per well. With a recombinant protein (positive control), you can use ~200 pg per well.
For limited cell numbers or primary cells, use the Protein Expression Sample Prep Kit. For larger amounts of cells (>50,000), or if you want to be able to normalize to total protein concentration, use the Protein Quant Sample Lysis Kit.
Software Data Analysis
You can, but it is not supported by our software (ProteinAssist™ Software). ProteinAssist™ Software is for relative quantitation only. You would have to use a third-party option, such as MasterPlex, to generate a standard curve and determine absolute quantitation. If you make a standard curve, you can use the NPC as a negative control. If needed you can also use mixed probes for your samples as a negative control. For AQ, you will also need to do a spike-in recovery and linearity tests to make sure the assay will give you accurate quantitative results.
For Research Use Only. Not for use in diagnostic procedures.