FlashPlate Kinase Assays
Overview
In the FlashPlate® format, kinase substrate is captured onto a FlashPlate, which contains scintillant embedded in the plastic of the plate. If the substrate has been phosphorylated by kinase, the transferred 33P- phosphate on the substrate will be in close enough proximity to the surface of the FlashPlate to interact with the scintillant, generating signal. Unreacted, radiolabeled ATP will be floating freely in solution, and will not be close enough to the surface of the plate to interact with the scintillant. This assay format is amenable to high-throughput screening and other medium- and high-throughput assays.
FlashPlate kinase assays
What do I need to run this assay?
- 33P-gamma-ATP (see table in next section)
- Substrate-of-interest, tagged if using affinity capture to FlashPlate
- Flashplate (pre-coated if using tagged substrate, or a Basic FlashPlate if you will be directly coating your substrate)
- Kinase
- High throughput radiometric detector (we recommend a TopCount® counter or MicroBeta® counter)
Products and catalog numbers
Radiolabeled ATP
- 33P-gamma-ATP is preferred over 32P-gamma-ATP because of its lower energy, leading to lower assay background
- EasyTides® products contain a dye in the buffer to aid with pipetting, and can be stored at 2-8°C
- NEG602K (high-throughput screening formulation) has a stabilizer in the buffer that keeps the product more stable at room temperature while setting up plates for high throughput screening.
| Compound | Specific activity (Ci/mmol) | Rad. conc. (mCi/mL) | Molar concentration (µM) | EasyTides version containing dye in buffer (Shipped ambient, store at 2-8°C) | Frozen version (Shipped on dry ice, store at -20°C) |
|---|---|---|---|---|---|
| ATP,[gamma-33P] | 3000 | 10 | 3.3 | NEG602H | NEG302H |
| ATP,[gamma-33P] for HTS (stabilized) | 3000 | 10 | 3.3 | NEG602K |
FlashPlates
| FlashPlate | Affinity | Well format | Number of plates | Catalog number |
|---|---|---|---|---|
| Basic | High-bind for direct coating | 96-well | 50 | SMP200001PK |
| 100 | SMP200J001PK | |||
| 200 | SMP200B001PK | |||
| 384-well | 5 | SMP400E001PK | ||
| 20 | SMP400001PK | |||
| 100 | SMP400J001PK | |||
| Streptavidin | Biotin | 96-well | 5 | SMP103001PK |
| 20 | SMP103A001PK | |||
| 100 | SMP103J001PK | |||
| 100 | SMP103J001PK | |||
| 384-well | 2 | SMP410001PK | ||
| 10 | SMP410A001PK | |||
| 100 | SMP410J001PK | |||
| 200 | SMP410B001PK |
Protocol-in-brief
Two different protocols are presented below. In the pre-coated substrate assay, your kinase substrate is first pre-coated to (or pre-incubated with) your plate, immobilizing the substrate to the plastic surface of the well. Then the enzyme is added to begin the kinase reaction. In the all-in-one assay, substrate and kinase are added together to the plate. If you will need to direct-coat your substrate to the plate (rather than using a biotinylated or otherwise tagged substrate to associate indirectly with a coated FlashPlate), you will need to use the first protocol. You may prefer to allow kinetics to happen in solution, rather than while the substrate is immobilized to the plate. Ideally, both formats should be tested if there is an option to choose between the two.
FlashPlate kinase assay workflow
Application notes and guides
- Application note: PKC and KDR kinase assays in 96-well format using a Basic FlashPlate (direct-coating)
- Application note: PI3 kinase assay in 96-well format using a Phospholipid-coated FlashPlate
- Application note: c-Src kinase assay in 384-well format using a Streptavidin-coated FlashPlate
- Application note: general information on converting from 96-well FlashPlate assays to 384-well FlashPlate assays
- Application note: compares a FlashPlate kinase assay to a LANCE Classic kinase assay and an AlphaScreen kinase assay
- FlashPlates knowledge base page
Citations
- Blackburn, C. et al. Discovery and optimization of N-acyl and N-aroylpyrazolines as B-Raf kinase inhibitors. Bioorg. Med. Chem. Lett 20, 4795-4799 (2010). Link
- Burgess, A. et al. Inhibition of S/G2 Phase CDK4 Reduces Mitotic Fidelity. Journal of Biological Chemistry 281, 9987 -9995 (2006). Link
- Prisic, S. et al. Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases. Proceedings of the National Academy of Sciences 107, 7521 -7526 (2010). Link
- Smith, C.K. et al. Expression and purification of phosphorylated and non-phosphorylated human MEK1. Protein Expr. Purif 52, 446-456 (2007). Link
- Sun, C. et al. High-Throughput Screening Assay for Identification of Small Molecule Inhibitors of Aurora2/STK15 Kinase. Journal of Biomolecular Screening 9, 391 -397 (2004). Link
Tips and FAQs
- For this particular FlashPlate assay, we advise washing the plate before reading to remove unreacted ATP from the well, in order to reduce assay background.
Other 爱游戏平台注册登录 kinase technologies
- LANCE Ultra TR-FRET kinase assays
- LANCE Classic TR-FRET kinase assays
- Alpha SureFire no-wash cellular kinase assays
- Alpha kinase assays
- DELFIA fluorescence kinase assays
Custom services at 爱游戏平台注册登录
爱游戏平台注册登录 offers custom radiochemicals, custom FlashPlates and barcoding, and custom assay development. If you are interested in custom services, please contact our custom teams: