Description
Farnesyltransferase Inhibitor Screening Kit (BA0250) (BA0250)
The Farnesyltransferase Inhibitor Screening Kit (SKU: BA0250) provides a convenient fluorimetric method for screening potential inhibitors of farnesyltransferase (FTase). FTase (EC 2.5.1.58) catalyses the transfer of a farnesyl group from farnesyl pyrophosphate to the cysteine residue at the C-terminus of target proteins; when not properly regulated, farnesylated proteins including the Ras superfamily of small GTPases can drive developmental disorders and cancer, making inhibitor screening valuable for oncology research. In this assay FTase reacts with farnesyl pyrophosphate and a dansyl-peptide substrate with measurable fluorescence at λem/ex = 340/550 nm, and inhibition is determined by the decrease in fluorescence. The non-radioactive, homogeneous mix-incubate-measure assay is supplied in a 400-test 384-well format, routinely achieves a Z'-factor of 0.8 or higher, and can be readily automated to assay thousands of samples per day. Lonafarnib is included as an IC50 control.
| Product Name: | Farnesyltransferase Inhibitor Screening Kit (BA0250) |
| SKU: | BA0250 |
| Detection Method: | Fluorimetric (λex/em = 340/550 nm) |
| Sample Type: | Farnesyltransferase enzyme and test compounds |
| Species Reactivity: | All |
| Assay Time: | 60 minutes |
| Kit Size: | 400 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20°C |
| Shelf Life: | 6 months |
| Shipping: | Gel Pack |
A fluorimetric inhibitor screening assay for farnesyltransferase (FTase). FTase reacts with farnesyl pyrophosphate and a dansyl-peptide substrate producing fluorescence at λem/ex = 340/550 nm; inhibition is measured as a decrease in fluorescence.
- Safe. Non-radioactive assay.
- Homogeneous and convenient. Mix-incubate-measure type assay. No wash and reagent transfer steps are involved.
- High-throughput. A Z'-factor of 0.8 and higher is routinely observed in a 384-well format. Can be readily automated to assay thousands of samples per day.
- High-throughput screening of FTase inhibitors and evaluation of drug modulators
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | This assay is based on an enzyme-catalysed kinetic reaction. To ensure identical incubation times, addition of Working Reagent should be quick and mixing brief but thorough; a multichannel pipettor is recommended. Note: FTase enzyme is not included in the kit. |
| 2 | Reagent preparation: use black, flat-bottom 384-well plates. Equilibrate all components to room temperature and briefly centrifuge tubes before opening. Prepare the Working Reagent (WR) fresh for each assay run. Prepare enzyme in buffer and use fresh (protocol optimised for recombinant rat FTase, Jena Bioscience cat# PR-102). Dissolve test compounds in a solvent of choice (e.g. DMSO); Lonafarnib is included as an IC50 control. |
| 3 | Enzyme and controls: transfer 5 µL of 37.2 µg/mL FTase (7.4 µg/mL final) into separate wells of a black 384-well plate. Reserve at least one well for no inhibitor (Control) and one with no enzyme (Blank); add 5 µL of 37.2 µg/mL FTase to the Control and 5 µL Assay Buffer to the Blank. Run at least duplicate reactions. |
| 4 | Add test compounds: to the enzyme wells add 5 µL of the test compounds. To the Control and Blank wells add 5 µL of the solvent used for the test compounds. Mix immediately and incubate 10 min at room temperature for test compounds to interact with FTase. |
| 5 | Prepare enough WR for all wells by mixing, per well: 0.5 µL Substrate, 20 µL Assay Buffer and 1 µL TCEP. Transfer 15 µL WR to all wells. Immediately tap the plate to mix and incubate at room temperature. |
| 6 | Read fluorescence intensity at 60 min at λex/em = 340/550 nm. |
FTase activity in the presence of test compounds: % Activity = (RFU_TestCpd – RFU_Blank) / (RFU_Control – RFU_Blank) × 100%, where RFU_TestCpd, RFU_Control and RFU_Blank are the fluorescence values of the test compound, no-inhibitor control and no-enzyme blank at 60 min.
| Component | Quantity | Storage |
| Assay Buffer | 12 mL | -20°C |
| Substrate | 200 µL | -20°C |
| 180 mM TCEP | 400 µL | -20°C |
| 10 mM Lonafarnib | 50 µL | -20°C |
