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View additional product information for Protein Expression Sample Prep Kit - FAQs (4405443)
16 product FAQs found
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
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.
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.
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.
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.
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.
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 .
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.
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.
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.
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.
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.
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.
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).
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.
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