Corning Epic System MAP Kinase
Corning® Epic® System:MAP Kinase
Authored by: Corning Incorporated Life Sciences
In this study, we used the Epic System to analyze the direct interactions between MAP kinase (MAPK), a serine threonine kinase with ~40kDa molecular weight (MW), and a set of small-molecule compounds with MWs from 315-362 Daltons (Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, USA). MAPK is involved in the control of cellular responses related to cytokines and stress. This kinase is known to be activated by a range of cellular stimuli like osmotic shock, lipopolysaccharide (LPS), inflammatory cytokines, UV light and growth factors. It is being considered as a major target for the development of anti-inflammatory drugs and inhibition of MAPK cascade.
Materials and Methods
The following experiments were performed to study the interactions between MAPK and small-molecule compounds:
- Immobilization of MAPK at different concentrations (12.5,25 and 50 μg/mL) in 20 mM HEPES buffer pH 7.0 and also in 20 mM MES buffer pH 6.0
- Monitoring of MAPK immobilization on Epic microplate and binding of five representative BIPI compounds
Baseline was created by reading with 10 mM Acetate buffer pH 5.5, 15 μL/well. Buffer was removed from each well. MAPK was immobilized for 1 hour by adding 15 μL/well MAPK on Epic microplate at different concentrations (12.5, 25 and 50 μg/mL) in 20 mM HEPES buffer pH 7.0 and also in 20 mM MES buffer pH 6.0
The Epic microplate was washed and blocked with 200 mM ethanolamine in 150 mM borate buffer pH 9.2 for 5 minutes. All wells were washed with assay buffer (50 mM HEPES pH 7.4, 50 mM KCl, 10 mM MgCl2, 0.05% CHAPS, 1% DMSO and added back 15 μL/well 10 mM Acetate pH 5.5. Final series of scans were taken on the instrument to measure the level of kinase immobilization on the Epic microplate. Buffer was removed from the Epic microplate and all wells were washed with assay buffer (50 mM HEPES pH 7.4, 50 mM KCl, 10 mM MgCl2, 0.05% CHAPS, 1% DMSO) twice. Finally, 15 μL of assay buffer with 1%DMSO was added to each well.
All small-molecule compounds were diluted at different concentrations using identical binding assay buffer. The final DMSO concentration was maintained at 1% in order to match the index of refraction of the binding assay buffer in the Epic microplate with immobilized MAPK.
MAPK immobilized the Epic microplate containing 15 μL binding assay buffer was read on the instrument to achieve the baseline. Next, 15 μL compound, in identical binding assay buffer, was added to the MAPK immobilized Epic microplate followed by mixing step. Final compound binding reads were taken by scanning the microplate continuously interrogating 16 wells at a time for predetermined number of scans. The kinase immobilization and compound binding levels were determined by subtracting the baseline readings from the final readings.
|Figure 3. At 1 μM, a compound with an apparent KD of 20 nM shows clear binding over a non-binding compound. Compound 1 (MW ~350 Dalton and apparent KD of 20 nM) binding to immobilized MAPK (~40 kDa) using the label-free Epic System (in blue), and the non-binder, compound 5 (in red), did not show the binding with MAPK.|
|Figure 4. MAPK was immobilized on an Epic® 384-well microplate and different amounts of each small-molecule compounds were added to obtain a saturation binding curve. The saturation binding curve derived KD values of different small molecules ranged from 57 nM to 1168 nM, which is consistent with the previously published data using the Biacore system.|
- The Corning® Epic® System has the ability to generate high quality information for small molecule and target protein interaction.
- Targets can be used without the need of labels (i.e., radiolabeling, fluorescent tags, etc.).
- The assay performed on the Epic System is robust and simple to run.
- The Epic System shortens the time it takes to progress from assay development to high throughput screening campaign.
- Apparent KD values for the small-molecule compounds (molecular weights from 315-362 Daltons) for binding to MAPK protein target (MW ~40 kDa) determined by the Epic System are comparable to Biacore data.
- Apparent KD values are reproducible with excellent CVs.
- ↑ S.J. Harper and P. LoGrasso, Cell Signal 2001;13(5):299-310
|Click [+] for other articles on||The Market Place for Lab Automation & Screening||Analytical Equipment, Measuring, Testing Detection Devices|