Here we show how the PerkinElmer EnSpire®Multimode Plate Reader with Corning®Epic®label-free technology can be used in 96-and 384-well microplate formats to non-invasively identify and characterize multiple G protein-coupled receptor (GPCR) pathways in living cells using an orthogonal approach. Furthermore, this orthogonal platform enables the use of both label-free and labeled technologies to comprehensively identify and characterize target and ligand behavior in both cell-based and biochemical assays, with greater confidence. By successfully monitoring the ligand-induced dynamic mass redistribution (DMR) in living cells and the ligand-dependent response interrogating protein:proteinand protein:smallmolecule biomolecularinteractions, we demonstrate that this label-free technology is a comprehensive and versatile tool for GPCR research enabling the generation of physiologically-relevant data.
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SBS 2011: Sensitive Cell-based and Biochemical Assays Using Epic(R) Label-free technology on the EnSpire(R) Multimode Plate Reader
1. Sensitive Cell-based And Biochemical Assays Using Epic ® Label-free
Technology On The EnSpire ® Multimode Plate Reader
Heidi Morgan, M.S., Janet Park-Bewsher, Paul Butler, Tim Cloutier, Ph.D.
1 Abstract 2 EnSpire Label-free Cell-Based Assays 3 EnSpire Label-free Biochemical Assays 4 EnSpire Label-free Is Both Sensitive
Here we show how the PerkinElmer EnSpire® To demonstrate that the EnSpire label-free platform yields Below, we use the EnSpire label-free instrument to analyze & Non-Invasive
comparable data to the Corning® Epic® label-free system and direct binding interactions between Carbonic Anhydrase II (30
Multimode Plate Reader with Corning® Epic® label-
provides a powerful complementary assay platform relative to kDa) and a set of small molecule compounds ranging from 157
free technology can be used in 96- and 384-well labeled technologies, we performed a comparative analysis to to 350 Daltons. The saturation binding curve KD values for all
microplate formats to non-invasively identify and the Epic® system and labeled technologies for multiple GPCR interactions are consistent with Epic label-free generated data
characterize multiple G protein-coupled receptor signaling pathways and protein:protein and protein:small and previously published studies (Myszka, D.G. Analysis of
(GPCR) pathways in living cells using an orthogonal molecule biomolecular interactions. Our results show the small-molecule interactions using Biacore S51 technology. Anal.
EnSpire label-free platform compares favorably to the Epic® Biochem. 329, 316-323 (2004).).These results indicate that the
approach. Furthermore, this orthogonal platform system in terms of sensitivity and detection, while EnSpire label-free system generates reproducible and high-
enables the use of both label-free and labeled simultaneously showing high correlation to published quality information for protein:small molecule interactions.
technologies to comprehensively identify and pharmacological and binding data with several labeled assays.
characterize target and ligand behavior in both cell- Molecular EnSpire EnSpire Epic Biacore**
based and biochemical assays, with greater Compound
Weight (Da) Z’ KD [nM] KD [nM] KD [nM]
confidence. By successfully monitoring the ligand- Benzenesulfonamide 157 0.72 880 Not Tested 848
induced dynamic mass redistribution (DMR) in living Carbachol 183 No Binding No Binding Not Tested Not Tested
cells and the ligand-dependent response 4-Carboxybenzene
interrogating protein:protein and protein:small sulfonamide (CBS)
201 0.52 615 700 893
Target Signal Sensitivity: Agonist The EnSpire label-free platform imposed
molecule biomolecular interactions, we demonstrate Acetazolamide 222 Not Tested 55 27 19 Activation Using the EnSpire Label-free no detrimental effects to cell viability
Platform used to calculate Z’. The subsequent to the label-free scans as
that this label-free technology is a comprehensive Dansylamide 250 Not Tested 701 513 760 sensitivity is such that partial and full demonstrated with the ATPlite®
and versatile tool for GPCR research enabling the Furosemide 330 0.51 787 550 513 agonists can be detected using label-free cytotoxicity assay. Experiment was
generation of physiologically-relevant data. (±)-Sulpiride 341 0.50 89* 29* 186*
technology as well as inverse and full conducted with A431 endogenous cells.
antagonists (data not shown).
*Concentration in µM
**Myszka, D.G. Analysis of small-molecule interactions using Biacore S51 technology. Anal. Biochem.
329, 316-323 (2004)
Human epidermoid carcinoma A431, Chinese Hamster Ovary The initial step in the biochemical assay protocol involves immobilization of
(CHO-K1), human astrocytoma 1321N1, and Human Embryonic the target onto the amine-coupling surface of the EnSpire label-free 6 The EnSpire Label-free Platform Offers:
biochemical microplate biosensor. (The reference area of the biosensor
Kidney 293 (HEK293) cells were used to test agonist and prevents non-specific target immobilization.) Next, the unbound target is
antagonist activity of either endogenously or recombinantly washed away followed by further equilibration of the biosensor and the initial
• More fully characterized information about both
expressed GPCR receptors, including Adrenergic-β2 (β2AR), baseline read. Finally, the analyte is added to the immobilized target enabling cellular and biochemical systems
Muscarinic 1 (M1), Opioid Mu (OP3), Purinergic P2Y11 and the final read. Relative to adjacent well referencing utilized by other label-free • Pathway-independent analysis & non-invasive, more
Adenosine A1, using the EnSpire with label-free technology and instruments, the EnSpire label-free platform self-referencing technology
several leading labeled cAMP and Ca2+ detection technologies. delivers data with lower variability and eliminates the need to assign physiologically relevant data
reference wells. • Ability to study difficult targets or weak biological
Cell-Based Assays interactions
Epic® technology measures • Highly sensitive, robust monitoring of direct
changes in light refraction
resulting from dynamic mass
protein:ligand binding events especially those weak
redistribution (DMR) within the interactions that would be difficult to detect using
cell which occurs in response to labeled assays.
receptor activation or
deactivation in a zone within the
• Direct monitoring of small molecule binding to an
cell’s monolayer. The change is immobilized protein with extremely high sensitivity.
indicated by a change in • Reduced cost and time required for assay
wavelength.
development for most targets by applying a label-free
strategy as described.
Biochemical Assays • Robust measurement of small molecule binding
Label-free biochemical assays ranging from 157 Da to 350 Da with KD values
measure changes in the index of
refraction upon a binding event. comparable to published Biacore® SPR data. This
As in cellular assays, the observation indicates that in situations where SPR
change is indicated by a shift in becomes a throughput bottleneck in the biophysical
wavelength.
process, use of the plate-based EnSpire with Epic®
label-free technology for studies based on KD prior to
SPR, will reduce the sample backlog of ka and kd ‘s
Agonist and antagonist dose-response curves and assay robustness for Gs, Gq and Gi leading to a more efficient workflow.
Clear and concentration-dependant dose response curves for each small molecule
pathways (β2AR , A1, Mu Opioid, M1) using EnSpire with label-free technology. binding to immobilized Carbonic Anhydrase II.
Corning and Epic are registered trademarks of Corning Incorporated. .
PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com