| Field | Specification |
|---|---|
| Assay Type | |
| Detection Instrument(s) | HTRF®-certified Microplate Reader (e.g. Tecan M1000, Tecan Spark) |
| Detection Method | |
| Product Type | |
| Shipping | |
| Storage |
Background
Tobacco Etch Virus protease (TEV protease) is a highly sequence specific cysteine protease. It has a strict 7 amino acid cleavage recognition sequence of Glu-Asn-Leu-Tyr-Phe-Gln ↓ (Gly/Ser). The high specificity makes this protease excellent for the removal of affinity-tags from purified recombinant proteins.
Assay Principle
The TEV Protease Activity Assay kit is a fluorogenic-based assay to measure TEV protease activity. The kit contains a TEV protease substrate that is labeled with fluorophore FAM and a quencher. Proteolytic activity of TEV protease cleaves the substrate and releases the FAM, resulting in the production of bright fluorescence which can be measured using a fluorescence reader at ex/em of 490 nM/520 nm. TEV protease activity then can be calculated in accordance with the fluorescence intensity. Purified TEV protease is included in the kit as a positive control.
Application
Measure TEV protease activity.
Instrument Required
A microplate reader capable of measuring fluorescence intensity is required. Aurora Biolabs, LLC; www.aurorabiolabs.com; San Diego, CA, USA. Tel: 858-215-4510 or 858-374-6010; Tech: 858-453-5700 58-453-5700
Kit Components
| Catalog No. | Item | Amount | Storage |
|---|---|---|---|
| 190001B | 25 mL | -20°C | |
| 96-well microplate, black | Room temperature |
Materials Not Supplied
- Microplate reader
- 0.5 M DTT
- Adjustable micro-pipettor
- Sterile Tips
Assay Protocol
- Step 1. Dilute 1 mM 5-FAM to 20 µM with the assay buffer prepared at step A (assay buffer A).
- Step 2. Make 2-fold series of dilutions with the assay buffer a to get 10, 5, 2.5 1.25, 0.625, 0.3125 and 0 µM solutions.
- Step 3. Aliquot 50 µL of the diluted solution to each well (96-well plate).
- Step 4. Dilute substrate solution 25-fold with the assay buffer A.
- Step 5. Add 50 µl of diluted substrate to each well.
- Step 6. Measure fluorescent intensity at excitation of 490 nm and emission of 520 nm.
- Step 7. Use the same machine settings when measure TEV protease activity afterwards. 5-FAM Standard y t i s n e t n I e c n e c s e r o u F l 10000 8000 6000 4000 2000 0 0 2000 4000 6000 8000 10000 FAM [nM] C. Measure TEV protease positive control activity
- Step 8. Thaw TEV protease protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: TEV protease protein is sensitive to freeze/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein.
- Step 9. Dilute the TEV protein 125-fold with the assay buffer A (from 1000 ng/µL to 16 ng/µL). Then, make a further dilution to 8, 4, 2, 0.5, 0 ng/µL.
- Step 10. Add 50 µl of diluted protein solution to each well (Test amount of the protein will be 400, 200, 100, 50, 25 and 0 ng per reaction).
- Step 11. Dilute substrate solution 25-fold with assay buffer A.
- Step 12. Add 50 µl of diluted substrate to each well.
- Step 13. Incubate at room temperature for 1 hour.
- Step 14. Measure fluorescent intensity at excitation of 490 nm and emission of 520 nm.
- Step 15. Plot fluorescent intensity versus protein concentration on a graph as below (subtract the average fluorescent intensity readings in the 0 ng wells from all of other wells to remove fluorescence background). TEV Activity y t i s n e t n I e c n e c s e r o u F l 7000 6000 5000 4000 3000 2000 1000 0 0 100 200 TEV [ng] 300 400 D. Measure TEV protease activity
- Step 16. Dilute TEV protease protein to 8, 4, 2, 0.5, 0 ng/µL with the assay buffer A.
- Step 17. Add 50 µl of diluted protein solution to each well (Test amount of the protein will be 400, 200, 100, 50, 25 and 0 ng per reaction). We recommend to run the reactions in duplicate.
- Step 18. Dilute substrate solution 25-fold with assay buffer A.
- Step 19. Add 50 µl of diluted substrate to each well.
- Step 20. Incubate at room temperature for 1 hour.
- Step 21. Measure fluorescent intensity at excitation of 490 nm and emission of 520 nm.
- Step 22. Plot fluorescent intensity versus protein concentration on a graph as below (subtract the average fluorescent intensity readings in the 0 ng wells from all of other wells to remove fluorescence background).
Biological Pathway / Process
Proteolysis (affinity tag removal; protein engineering)
Therapeutic / Disease Area
General / Biotechnology
▶▼What activity does the TEV Protease Activity Assay Kit measure?
This kit measures TEV Protease (Tobacco Etch Virus cysteine protease) enzymatic activity using a fluorescence-based format. The assay is designed for cell-free, homogeneous conditions that support both endpoint and kinetic measurements, and is suitable for IC₅₀ determination of inhibitor candidates.
▶▼What instrument or plate reader is required?
A fluorescence microplate reader is required to read this assay. The specific excitation and emission wavelengths depend on the detection mode used. Please refer to the product datasheet for exact instrument settings and compatible reader models.
▶▼How many reactions are included, and is bulk ordering available?
This kit is available in 96 reactions. Each reaction is conducted in a 96-well / fluorescence plate reader format, making it directly compatible with standard liquid-handling robotics for HTS applications. For bulk orders or custom quantities, please contact BioHippo or submit a quote request — distributor pricing is available.
▶▼Has the assay performance been validated?
Yes. Aurora Biolabs validates each kit using reference compounds to confirm assay window, signal-to-background ratio, and reproducibility prior to release. Specific validation data are provided in the product datasheet. Users are encouraged to determine the Z′ factor under their own experimental conditions.
▶▼What are the storage and shipping requirements?
The kit ships on Dry Ice and should be stored at -80°C upon receipt. Individual components may have different storage requirements — please refer to the component table in the datasheet. Protein components are sensitive to freeze–thaw cycles; aliquot on first thaw and avoid repeated freeze–thaw.
Need a different assay kit format or extra support beyond the catalog item? We offer customization and add-on services for assay kits to better fit your target, workflow, and detection platform. Options may include assay format customization (biochemical, binding, or cell-based), detection method selection such as TR-FRET, fluorescence polarization, luminescence, or colorimetric readout, target-specific reagent development including recombinant proteins, conjugates, antibodies, substrates, tracers, and controls, as well as protocol optimization for sensitivity, specificity, signal window, and reproducibility. We can also support kit component adjustments, plate format and scale options, QC and validation packages, bulk or custom packaging, and related assay development services when a standard kit does not fully meet your needs. Click Talk to a Scientist to submit a request, email us at support@biohippo.com, or explore our Research Services for additional support. Our team will be in contact with you shortly.
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