Description
Farnesyltransferase Activity Assay Kit (BA0241) (BA0241)
The Farnesyltransferase Activity Assay Kit (SKU: BA0241) provides a convenient, non-radioactive fluorimetric method for assaying farnesyltransferase (FTase, EC 2.5.1.58) activity in biological samples. FTase 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 lead to developmental disorders and cancer, making simple high-throughput activity assays valuable for cancer research. In this assay FTase reacts with farnesyl pyrophosphate and a dansyl-peptide substrate, releasing a product measurable by fluorescence at λem/ex = 340/550 nm. The 'mix-incubate-measure' format requires no wash or reagent-transfer steps and can be automated to assay thousands of samples per day. Note that the FTase enzyme is not included in the kit.
| Product Name: | Farnesyltransferase Activity Assay Kit (BA0241) |
| SKU: | BA0241 |
| Detection Method: | Fluorimetric (FL340/550 nm) |
| Detection Range: | 0.024 - 3.2 U/L FTase (384-well plate) |
| Sample Type: | ['Biological samples', 'Purified enzyme'] |
| Species Reactivity: | All |
| Assay Time: | 60 min |
| Kit Size: | 400 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20°C |
| Shelf Life: | 6 months |
| Shipping: | Gel Pack |
A fluorimetric high-throughput kit for the quantitative determination of farnesyltransferase (FTase) enzyme activity in biological samples and purified enzyme. FTase reacts with farnesyl pyrophosphate and a dansyl-peptide substrate to release a fluorescent product measured at λem/ex = 340/550 nm, with a linear detection range of 0.024 - 3.2 U/L in a 384-well plate.
- Safe and convenient. Non-radioactive, 'mix-incubate-measure' type assay with no wash or reagent-transfer steps.
- Sensitive and accurate. Linear detection range 0.024 - 3.2 U/L FTase in a 384-well plate assay.
- High-throughput. Can be readily automated to assay thousands of samples per day.
- For quantitative determination of FTase enzyme activity in biological samples.
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 | Prior to assay, equilibrate all components to room temperature and briefly centrifuge tubes before opening. Use black flat-bottom 384-well plates and prepare Working Reagent fresh for each run. Prepare enzyme in buffer and use fresh; experimentally determine the optimal amount of enzyme per well (FTase enzyme is not included). |
| 2 | Transfer 5 µL of the samples to separate wells. |
| 3 | Prepare enough Working Reagent for all sample wells by mixing, per well, 0.5 µL Substrate, 30 µL Assay Buffer and 1 µL TCEP. Add 25 µL Working Reagent to all sample wells and immediately tap the plate to mix. |
| 4 | Read fluorescence intensity at time zero and at 60 min at λex/em = 340/550 nm, or read fluorescence kinetically for 60 minutes. |
FTase Activity = (Reaction Vol / Sample Vol) × [(F60 – F0) / ((4.3·F0 – F0)/[Substrate]·t)] × n = (Reaction Vol / Sample Vol) × [(F60 – F0)/F0] × 0.303 × n (U/L), where F60 and F0 are the fluorescence intensities at 60 and 0 min; [Substrate], Reaction Vol and Sample Vol are 10 µM, 30 µL and 5 µL respectively; t is the reaction time (60 min); 4.3 is the conversion factor for maximum sample fluorescence at 6 hours; and n is the sample dilution factor. Unit definition: one unit of FTase catalyses the transfer of 1 µmole of the farnesyl group per minute under the assay conditions.
| Component | Quantity | Storage |
| Assay Buffer | 12 mL | -20°C |
| Substrate | 200 µL | -20°C |
| 180 mM TCEP | 400 µL | -20°C |
