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Applied Biosystems™ TaqMan™ Protein Assays Open Kit

Catalog No. 4453745
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Catalog No. 4453745 Supplier Applied Biosystems™ Supplier No. 4453745

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Description

Sensitive Detection — directly measure in vitro protein-protein interactions with only a few thousand cells.

  • Small Sample Size - use up to 500 times less sample input compared to traditional methods
  • Sensitive Detection - directly measure in vitro protein-protein interactions with only a few thousand cells
  • Revolutionary - directly correlate mRNA and/or microRNA to protein
  • Simple Workflow - 1-step lysis sample preparation, universal protocol
  • Quantitative - protein marker expression fold changes detected

Specifications

Content And Storage The TaqMan™ Protein Assays Open Kit contains the TaqMan™ Protein Assays Oligo Probe Kit (4448549) and the TaqMan™ Protein Assays Buffer Kit (4448571)for making TaqMan™ Protein Assay probes.
Detection Method Primer-probe
GC-Rich PCR Performance Low
PCR Method qPCR
Reaction Speed Fast
Technique Proximity Ligation Assay
For Use With (Equipment) 7500 Fast System, 7500 System, 7900HT Fast System, 7900HT System, StepOnePlus™, Fast Mode
Label or Dye FAM
Passive Reference Dye ROX (Pre-mixed)
Product Line TaqMan
Product Type Protein Assays Open Kit
Quantity 4,000 reactions
Target Specificity Not Target-Specific
Hot Start No
No. of Reactions 4000 Reactions
Sample Type Tissue, Cells
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Frequently Asked Questions (FAQs)

Why do I not see any difference in expression between my experimental samples and the negative control in my TaqMan Protein Assay?

There may be several reasons for not seeing any change in expression. Check for the following:

- Did the antibody probes pass the forced proximity test?
- What is being used for the negative control? If there is endogenous expression of the target, consider using buffer instead of a “negative” lysate.
- Make sure to test a range of inputs, using 2- to 3-fold dilutions
- For cells: 1-500 cells
- For recombinant protein: ~200 pg/well
- For total protein (tissue): 1-1,000 ng
- For cell lysate: ~50 ng total protein/well
- Make sure to use a positive control to ensure detection
- Test with one of the control assays, such as for hCSTB or hICAM1

In a TaqMan Protein Assay, what can I do if my antibody probe does not pass the forced proximity test?

The forced proximity test helps to assess the quality of the biotinylated antibody. If it does not pass, then either the antibody is under-biotinylated, or there still is free biotin in the solution. Even as little as 80 nM free biotin can cause the test to fail. Make sure to dialyze the antibody well (change the buffer at least five times, including one overnight exchange). We recommend using the Slide-A-Lyzer Mini Dialysis Unit. Repeat the dialysis steps and perform the test again.

How do I analyze the TaqMan Protein Assay data?

ProteinAssist Software is free analysis software that can be used for relative quantitation analysis of your data, directly compatible with the data from most real-time instruments.

Can I use the TaqMan Protein Assays for absolute quantitation (AQ)?

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.

What is an NPC, and what can I use for one?

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.

What instruments are compatible with TaqMan Protein Assays?

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), ViiA7 (v1.0 and later) systems. If you have a different system, please consult techsupport@thermofisher.com for more information.

What products do I need to start using a TaqMan Protein Assay?

Check out our TaqMan Protein Assays Product Overview (https://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/nucleic-acid-amplification-expression-profiling/pdfs.par.74892.file.dat/taqman%20protein%20assays%20.pdf) for a complete explanation and list to help you get started .

Which is better, a monoclonal or a polyclonal antibody for a TaqMan Protein Assay?

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.

Do I have to use a biotinylated antibody for a TaqMan Protein Assay? What if I can't find one?

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 non-biotinylated antibody and a biotinylation kit, or utilize our custom services (https://www.thermofisher.com/us/en/home/products-and-services/services/custom-services/assay-development-services/custom-antibody-and-protein-labeling.html) to have it done for you.

How do I biotinylate my antibody for a TaqMan Protein Assay?

In some cases, you may have to biotinylate the antibody yourself using a commercial kit. Please follow the guidelines in the Probe Development Protocol (https://tools.thermofisher.com/content/sfs/manuals/cms_083917.pdf, 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.

How do I choose an antibody for a TaqMan Protein Assay?

Please follow the guidelines in the Protein Assay Chemistry Guide (https://tools.thermofisher.com/content/sfs/manuals/cms_071273.pdf, ~p. 43) for help in chhosing an antibody.

Which antibodies have you tested in a TaqMan Protein Assay?

Here is our current list of pre-tested antibodies (https://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/nucleic-acid-amplification-expression-profiling/pdfs.par.15243.file.dat/list-of-screened-antibodies-openkit.pdf). If your target is not on this list, you can use your own antibody using the TaqMan Protein Assays Open Kit.

How does a TaqMan Protein Assay compare to an ELISA for sensitivity?

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.

What distance is spanned by the TaqMan Protein Assay probes?

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).

What is the largest protein you have studied using the TaqMan Protein Assays?

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.

How is the TaqMan Protein Expression Assay different from an ELISA (enzyme-linked immunosorbent assay)?

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

For Research Use Only. Not for use in diagnostic procedures.