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Label-Free Detection FAQs

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General

• What are the main features of the Epic® System?

The main features of the Epic® System are its ability to perform label-free biochemical and cell-based assays in a 384-well microplate format.  It is easily integrated with existing automation and can reach throughputs up to 40,000 wells in 8 hours.  Generally, Epic® assays have reduced assay development times and enable researchers to obtain more physiologically relevant data.

• What assay classes have been demonstrated on the Epic System?

Epic assays are applicable across a wide-variety of biochemical and cell-based assays including small molecule-protein binding, functional protease, kinase, protein-protein interactions, GPCRs, viral detection, and ion channels.  For a complete list of Epic applications, please see our technical library.

• What is the role of liquid handling in Epic assay performance?

Liquid handling parameters are key factors in developing and can impact an Epic assay variability and robustness (Z').  Recommended starting set points for z-height, dispense/aspirate speeds and volumes, and number of mixes for both biochemical and cell-based assays can be found in the standard assay standard operating procedures (SOPs).  Corning Field Application Scientists can provide training on optimizing liquid handling parameters.

• What service and support does Corning offer for the Epic System?

Assay support is provided by Corning's team of Field Application Scientists (FAS), who are based in North America, Europe and Japan. In addition, Corning has a team of Field Service Engineers (FSE) to provide knowledgeable service on Epic instruments, worldwide. Corning has two Applications Centers where ongoing work is done on Epic application development; these labs are located in Corning, New York, and close to Paris in Fountainebleau, France.

• Can I coat Epic® plates myself?

Yes. Cell-based assay microplates (catalog # 5040) can be used for "do-it-yourself" coatings. Please contact your local Field Application Scientist for specific details.  Biochemical assay plates are not available at this time for do-it-yourself coating.

• How do I know that the signal I measure an Epic assay is specific?

As with other assays, specificity in Epic assays is determined by the use of appropriate positive and negative controls, and experiments designed to in answer the following questions:  Is the response dose-dependent? Is it saturable?  Can it be inhibited?

• What are the advantages of Epic assays compared to traditional (fluorescence, luminescence, radiolabeled) assays?

Unlike fluorescence, luminescence and radiolabeled assays, Epic assays avoid the time and expense associated with labeling. The use of fluorescent or radioactive labels not only requires a priori knowledge of targets and their natural ligands, but has been known to cause undesirable and unanticipated interactions that can compromise screening data and lead to false conclusions.  Additionally, Epic label-free assays enable researchers to use non-engineered cell lines and primary cells ensuring a more physiologically relevant response.

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Instrumentation

• Can I choose specific portions of the sensor where the system will scan?

No. Standard plate scanning algorithms are fixed with respect to the regions where they scan.  In case of technical difficulty, please contact your local Field Application Scientist who can provide additional trouble-shooting support.

• Can I use the LHA to wash plates (instead of using a stand-alone washer)?

Although pipettors similar to the LHA can be used to wash plates, we recommend that a dedicated plate washer be used for optimal results.

• What flexibility do I have in running the LHA?

The Epic Quest software enables you to design and perform custom process flows.  The method management system provides pre-defined assay step types and parameters that can be ordered and modified. The newly created custom process flow will automatically be stored and can be recalled or exported to other Epic Systems.

• Can I integrate Epic with other devices such as robotic arms, incubators, etc?

Yes.  The Epic System is designed to be an integrated device in an HTS workstation.  It has been integrated into numerous workstations provided by third party vendors.  Email us at epic@corning.com to discuss your specific integration needs.  We can help define your needs and work with third party vendors to ensure seamless integration.

• How many plates can be run on the LHA?

The system can run a total of 20 plates at a given time.  The total plate quantity can be a mix of Epic plates and compound plates.

• Can I use the LHA as a standalone pipettor?

Yes.  Customers can operate the LHA as a standalone pipettor using the CyBio Composer software to control the CyBi-Well.  However, it cannot be used as a stand-alone pipettor while running assays on Epic.

• Can the Epic + LHA execute parallel processes?

Yes, the Epic + LHA can execute parallel processes.  However, one cannot run the pipettor separately while the instrument is reading.

• What is the Liquid Handling Accessory (LHA)?

The LHA is an integrated accessory that provides automated plate loading with a pipettor on an Epic System.  It converts the Epic Reader into an assay development workstation with a capacity of 20 microplates with lids.

• Can I interleave plates on the LHA?

The Epic software will automatically search for interleaving opportunities and optimize plate handling, if possible.  Interleaving cannot be controlled manually.  Contact Corning Sales or Service to discuss your specific interleaving needs.

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Cell-Based Assays

• What is DMR?

DMR stands for Dynamic Mass Redistribution and refers to the movement of intracellular material in response to a stimulus.

• What surfaces are available for cell assays?

Two surfaces are commercially available: uncoated cell surface (catalog # 5040) and fibronectin-coated (catalog # 5042).

• Is self-referencing used in cell-based assay plates?

No.  Because the assay signals in cell-based assays are generally large (>~100pm), self-referencing is not required.

• Can I test multiple cell lines on a single plate?

Yes.  We have successfully cultured two different cell lines in adjacent columns on a single plate and tested their response to various GPCR agonists.

• What are the important parameters to optimize in a cell assay?

The most important parameters to be optimized for each new cell assay are: 1) cell seeding density; 2) plate type (uncoated or fibronectin-coated); 3) culture conditions; 4) liquid handling parameters for compound dispensing.

• How do I choose the correct plate for a cell-based assay?

For loosely-adherent cells, we recommend fibronectin-coated plates (catalog # 5042).  For strongly adherent cells and suspension cells, we recommend that you evaluate both fibronectin-coated and uncoated plates (catalog #5040). 

• What is the minimum receptor expression level for which a reliable signal can be detected?

The minimum receptor expression level for which a reliable signal can be detected is dependent upon the cell line, the specific receptor biology, and the signaling network; for GPCRs, the minimum level is on the order of 1000 - 3000 copies per cell

• Can I detect the direct binding of a ligand to a cell? What do I measure in a cell-based assay?

In cell based assays, the Epic System does not measure the direct binding of a ligand to a cell.  The Epic System measures the downstream, integrated response of the cell to the binding/activation of the biological target within ~150nm from the sensor surface.

• Can the Epic® System be used to study suspension cells?

Yes.  The Epic® System can be used to study both adherent and non-adherent (suspension) cells.

• Can I directly use frozen cells with the Epic System?

Yes.  Frozen cells can be directly seeded in Epic microplates.

• Can Epic microplates be used with an optical microscope?

Yes.  The bottom of the Epic microplate is transparent and can be used with optical microscopes.

• Are Epic cell assays sensitive to cell density?

Yes. In general we recommend running assays with a confluent monolayer.  For both adherent and non-adherent cells, the optimal seeding density is something that should be optimized for each assay.

• Does the Epic signal depend upon the expression level of the receptor?

Yes. However, for a given cell type, there is a maximal signal one can obtain for a particular receptor. The expression threshold required to reach the maximal signal is dependent on (1) the receptor; (2) the cell type; (3) the coupling efficiency to G protein; (4) the ligand; (5) the signaling capacity of the receptor; and (6) the interaction of the receptor with other proteins.

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Biochemical Assays

• Can I capture biotinylated proteins through immobilized streptavidin?

Yes.  Biotinylated peptides, proteins, and nucleic acids can be immobilized via streptavidin capture.  Detailed protocols are available for streptavidin immobilization.  As with any immobilization, we recommend that the pH of the capture buffer be optimized for each biotinylated protein.  For some biotinylated proteins we have observed that capturing efficiency is highest when using a pH below the isoelectric point of the protein.  For nucleic acids and small peptides, physiological pH works well.

• Why don't I observe any immobilization of my protein?

Suboptimal immobilization pH or insufficient protein concentration are the most common causes of low protein immobilization.  For any new protein target, we recommend testing immobilization pH values of 0.5 and 1 unit below the isoelectric point of the protein.  Generally, a protein concentration of 50ug/mL works well, but for some proteins, higher concentrations may be required.

• What are the advantages of the self-referencing technology in biochemical assay plates?

Relative to adjacent well referencing, self-referencing technology delivers data with lower variability and minimizes the number of additional reference wells required.

• What is the compound molecular weight detection limit for biochemical assays?

The smallest molecular weight compound that can be detected is a function of the size of the immobilized protein.  For a 30kDa protein, the smallest analyte we have detected is ~150Da.

• What is a basic assay flow for a biochemical assay?

The basic assay flow for biochemical assays is 1) target immobilization; (1 hour to overnight); 2) wash to remove unbound protein; 3) sensor equilibration; 4) baseline read; 5) analyte addition; 6) final read.

• How much protein is required for protein immobilization?

The amount of protein required for immobilization is somewhat protein dependent, but good results can generally be obtained using a concentration of 50ug/mL and a volume of 10-15uL/well.

• How much thermal equilibration time is required for biochemical assays?

For high sensitivity measurements (e.g. signal levels < ~50pm) we recommend at least a 20min equilibration time in the instrument.

• What is the correlation between the Epic response and mass density?

As a general guideline, 1pm of response corresponds to a protein density of ~3-5pg/mm2

• Can I use Epic to determine stoichiometry?

Yes.  In a biochemical assay, it is possible to determine if the binding signal levels are consistent with 1:1 binding versus a higher stoichiometry.  By measuring the amount of protein immobilized, one can calculate a theoretical maximum binding signal for an analyte.  If the observed signal in an assay is significantly larger than the maximum theoretical signal, this could indicate either greater than 1:1 binding stoichiometry or compound aggregation.

• Can the Epic System be used to determine kinetic constants of ligand binding?

Because the Epic System does not utilize flow cells, measurements of kinetics (kon, koff) will be diffusion-limited.

• Can the Epic System be used to identify promiscuous or aggregating compounds?

Yes.  In a biochemical assay, it is possible to determine if the binding signal levels are consistent with 1:1 binding versus a higher stoichiometry.  By measuring the amount of protein immobilized, one can calculate a theoretical maximum binding signal for an analyte.  If the observed signal in an assay is significantly larger than the maximum theoretical signal, this could indicate either greater than 1:1 binding stoichiometry or compound aggregation.

• Can I use part of an Epic biochemical assay plate?

In general we do not recommend using half of the plate with the intent to use the other half at a later time. Biochemical assay plates (catalog # 5041) are coated with a pre-activated surface chemistry that has an open pouch life of ~8 hours.

• Can the Epic System be used with complex/crude samples?

Yes.  We have successfully performed biochemical assays with samples in serum and cell lysates.  For cell-based assays we have successfully performed experiments in culture media.