Label Free Detection FAQs

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[{"resourcePath":"/au/en/products/life-sciences/resources.html","faqCategory":"Instrumentation","faqList":[{"answer":"Epic technology is available in the Corning Epic Gen I System, the Corning Epic BT System, as well as in the PerkinElmer® EnSpire® Multimode Plate Reader. For more information regarding the EnSpire, please visit <a adhocenable=\"false\" href=\"\" target=\"_blank\"></a>. The Epic Gen I System is available with on-board liquid handling and internal microplate storage in a temperature controlled environment. The Epic BT System is a small, bench-top sized instrument with the ability to run assays at 37°C by placing the instrument inside a non-humidified incubator. The optical biosensor technology remains the same between the two systems, though the scanning algorithms and data collection methods differ slightly. For additional details please contact your local Drug Discovery Specialist or Field Applications Scientist .","question":"What types of Epic readers are available?"},{"answer":"Yes. Both the Gen I Epic System, and the Epic BT System can be integrated into HTS workstations provided by third party vendors. Contact your local sales representative or Field Application Scientist to discuss your specific integration needs. We can help define your needs and work with third party vendors to ensure seamless integration.","question":"Can I integrate Epic with other devices such as robotic arms, incubators, etc.?"},{"answer":"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.","question":"Can I choose specific portions of the sensor where the system will scan?"},{"answer":"Yes. On the Gen I Epic System in Epic Quest software, parameters can be set to collect a specified number of data points, or over a specified range of time, or via both time and frequency. By modifying the total duration of time and number of data points collected, the scan time can be optimized. If just time, or just data points are selected the instrument will scan at a maximum throughput rate. The Epic BT System has an update rate of 3 seconds and can be altered to collect data at a slower rate by averaging data points together. Please contact your local Field Applications Scientist who can provide additional support. ","question":"Can I modify the update rate for scanning the sensor?"},{"answer":"The Gen I Epic system can run a total of 20 microplates at a given time. The total plate quantity can be a mix of Epic microplates and compound source microplates.","question":"How many microplates can be run on the Gen I Epic System?"},{"answer":"The LHA is an integrated accessory for the Gen I Epic System that provides automated plate loading with a pipettor. The LHA converts the Epic Reader into an assay development workstation with a capacity of 20 microplates with lids.","question":"What is the Liquid Handling Accessory (LHA)?"},{"answer":"Yes, buffer exchange protocols can be written to wash 384-well Epic microplates using the LHA on the Gen I Epic System. Please contact your local Field Applications Scientist for support.","question":"Can I use the Liquid Handling Accessory (LHA) on the Gen I Epic System to wash microplates (instead of using a stand-alone washer)?"},{"answer":"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. Custom process flows can be saved, recalled or exported to other Epic Gen I Systems.","question":"What flexibility do I have in running the Liquid Handling Accessory (LHA)?"},{"answer":"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 Gen I System.","question":"Can I use the Gen I Epic System LHA as a standalone pipettor?"},{"answer":"Yes. However, the pipettor cannot be run separately while the Epic Gen I System is running an assay.","question":"Can the Gen I Epic System + LHA execute parallel processes?"},{"answer":"Interleaving is an option when setting up methods in Epic Quest software. Once checked, the Epic software will automatically search for interleaving opportunities and optimize plate handling, if possible. Interleaving cannot be controlled manually. Contact your local Field Applications Scientist to discuss your specific interleaving needs.","question":"Can I interleave microplates with the Epic Gen I System?"}]},{"resourcePath":"/au/en/products/life-sciences/resources.html","faqCategory":"Cell-based Assays","faqList":[{"answer":"Two surfaces are commercially available: uncoated and fibronectin-coated cell assay microplates.","question":"What surfaces are available for cell-based assays?"},{"answer":"In cell based assays Epic technology does not measure the direct binding of ligand to receptor. The technology measures the downstream, integrated physiological response of the cell to the binding/activation of the biological target within a sensing region of ~150 nm from the sensor surface. We refer to the movement of intracellular components in response to a cellular stimulus, the Dynamic Mass Redistribution (DMR). Responses are measured as wavelength shifts (units of pm) in the reflected light from the bottom of the optical biosensor. ","question":"Can I detect the direct binding of a ligand to a cell? What do I measure in a cell-based assay?"},{"answer":"DMR stands for Dynamic Mass Redistribution and refers to the movement of intracellular components in response to a cellular stimulus.","question":"What is DMR?"},{"answer":"Yes, as long as the cell culture conditions are similar. We have successfully cultured multiple cell lines in adjacent columns on a single plate and tested their response to various stimuli.","question":"Can I test multiple cell lines on a single plate?"},{"answer":"The most important parameters to optimize for each new cell assay are: 1) cell seeding density; 2) microplate type (uncoated, fibronectin-coated, or self-coated); 3) culture conditions; 4) liquid handling parameters for compound dispensing and mixing.","question":"What are the important parameters to optimize in a cell-based assay?"},{"answer":"For loosely-adherent cells, we recommend fibronectin-coated microplates. For strongly adherent cells and suspension cells, we recommend that you evaluate both fibronectin-coated and uncoated microplates. ","question":"How do I choose the correct plate for a cell-based assay?"},{"answer":"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.","question":"What is the minimum receptor expression level for which a reliable signal can be detected?"},{"answer":"Yes. Epic Technology has successfully been used to study both adherent and non-adherent (suspension) cells. Liquid handling parameters for compound addition may require optimization when working with suspension cells.","question":"Can Epic Technology be used to study suspension cells?"},{"answer":"Yes. Frozen cells can be directly seeded into Epic microplates.","question":"Can I directly use frozen cells?"},{"answer":"Yes. The bottom of the Epic microplate is transparent and can be used with optical microscopes.","question":"Can Epic microplates be used with an optical microscope?"},{"answer":"Yes. In general we recommend running assays with a confluent monolayer. For both adherent and non-adherent cells, the optimal seeding density should be empirically determined during assay optimization.","question":"Are Epic cell-based assays sensitive to cell density?"},{"answer":"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 ligand; (4) the signaling capacity of the receptor; and (5) the interaction of the receptor with other proteins.","question":"Does the Epic signal depend upon the expression level of the receptor?"},{"answer":"Yes. As the Epic response is sensitive to mass movement within a sensing region of ~150 nm from the bottom of the sensor, mass degradation due to cytotoxic events or viral lysis will result in large negative responses. ","question":"Can Epic Technology be used for cytotoxicity or viral lysis assays?"},{"answer":"Yes. Through the use of receptor, kinase, and downstream signaling component inhibitors, specifics detailing the signaling pathway biology can be identified.","question":"Can downstream cellular signaling pathway biology be revealed with Epic Technology?"},{"answer":"The Epic BT System can placed inside a non-humidified incubator for running assays at 37°C.","question":"Can cell-based assays be performed at 37°C?"}]},{"resourcePath":"/au/en/products/life-sciences/resources.html","faqCategory":"Biochemical Assays","faqList":[{"answer":"Biochemical assay microplates are available in three formats: standard biochemical, enhanced biochemical, and streptavidin-coated. ","question":"What surfaces are available for biochemical assays?"},{"answer":"Yes. Biotinylated peptides, proteins, and nucleic acids can be captured via immobilized streptavidin. Protocols are available for streptavidin immobilization on standard biochemical assay microplates. Commercially available streptavidin-coated microplates are also available. ","question":"Can I capture biotinylated proteins through immobilized streptavidin?"},{"answer":"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 across a range of pH values. For some proteins, we have observed that capturing efficiency is highest when using a pH of 0.5 and 1 unit below the isoelectric point of the protein. Generally, a protein concentration of 50 µg/mL works well, but for some proteins, higher concentrations may be required.","question":"Why don't I observe any immobilization of my protein?"},{"answer":"Both the standard and enhanced Epic biochemical microplates immobilize target proteins via primary amine coupling. The enhanced microplates are user-activated and provide enhanced sensitivity, particularly for assays with very small response windows. The enhanced Epic microplates contain a spacer arm chemistry which may enable better positioning and presentation of the immobilized protein for binding site availability.","question":"What is the difference between the Epic standard and enhanced biochemical microplates?"},{"answer":"Relative to adjacent well referencing, self-referencing technology delivers data with lower variability and minimizes the number of additional reference wells required.","question":"What are the advantages of the self-referencing technology in biochemical assay microplates?"},{"answer":"The smallest molecular weight compound that can be detected is a function of the size of the immobilized protein and ratio of binding. For a 30 kDa protein, the smallest analyte we have detected binding in a 1:1 ratio is ~150 Da. The larger the immobilized protein, the larger the analyte must be to detect binding.","question":"What is the compound molecular weight detection limit for biochemical assays?"},{"answer":"The basic assay flow for a biochemical assay is 1) target immobilization (1 hour to overnight); 2) wash to remove unbound protein; 3) sensor equilibration; 4) baseline read; 5) analyte addition and mixing; 6) final read.","question":"What is a basic assay flow for a biochemical assay?"},{"answer":"The amount of protein required for immobilization is somewhat protein dependent, but good results can generally be obtained using a concentration of 50 µg/mL.","question":"How much protein is required for protein immobilization?"},{"answer":"We recommend at least a 20 min equilibration time at the temperature of the Epic instrument prior to initiating baseline acquisition.","question":"How much thermal equilibration time is required for biochemical assays?"},{"answer":"As a general guideline, 1 pm of response corresponds to a protein density of ~3-5 pg/mm<sup>2</sup>","question":"What is the correlation between the Epic response and mass density?"},{"answer":"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.","question":"Can I use Epic to determine stoichiometry?"},{"answer":"Because Epic Technology does not utilize flow cells, measurements of kinetics (kon, koff) will be diffusion-limited.","question":"Can Epic Technology be used to determine kinetic constants of ligand binding?"},{"answer":"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.","question":"Can Epic Technology be used to identify promiscuous or aggregating compounds?"},{"answer":"In general we do not recommend using a portion of the microplate with the intent to use the remainder at a later time. Biochemical assay microplates are coated with a pre-activated surface chemistry that has an open pouch life of ~8 hours.","question":"Can I use part of an Epic biochemical assay microplate?"}]},{"resourcePath":"/au/en/products/life-sciences/resources.html","faqCategory":"Label-free Detection FAQs","faqList":[{"answer":"Epic is a broad utility platform label-free optical biosensor technology used for cell-based, biochemical, and compound aggregation assays. The Gen I Epic System is compatible with 384-well and 1536-well Epic microplate formats, while the Epic BT System is compatible with 96-well, 384-well, and 1536-well Epic microplates. Both systems can be integrated with existing automation for high-throughput screening. Epic Technology is also available in the EnSpire® Multimode Plate Reader available from PerkinElmer®. Generally, Epic assays have reduced assay development times and enabled researchers to obtain more physiologically relevant data.","question":"What is Epic®?"},{"answer":"Epic Technology is applicable across a wide-variety of biochemical assays including small molecule-protein binding, functional protease, kinase, protein-protein interactions, and compound aggregation. GPCRs, RTKs, and ion channels are the most common assay classes for cell-based assays in both cell line and primary cell types. For a complete list of Epic applications, please see our technical library.","question":"What assay classes have been demonstrated using Epic Technology?"},{"answer":"Liquid handling parameters can impact maximum assay response, 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 on-site training for optimizing liquid handling parameters.","question":"What is the role of liquid handling in Epic assay performance?"},{"answer":"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 also has an Applications Center in Kennebunk, Maine where ongoing work is done on Epic application development.","question":"What service and support does Corning offer for Epic Technology?"},{"answer":"Corning offers cell-based assay microplates pre-coated with fibronectin in 96-well, 384-well, and 1536-well Epic microplate formats. However, uncoated cell-based assay microplates can be used for &quot;do-it-yourself&quot; coatings. Please contact your local Drug Discovery Specialist or Field Application Scientist for specific details. ","question":"Can I coat Epic microplates myself?"},{"answer":"As with other assays, specificity in Epic assays is determined by the use of appropriate positive and negative controls, and experiments designed to answer the following questions: Is the response dose-dependent? Is it saturable? Can it be inhibited?","question":"How do I know that the signal I measure in an Epic assay is specific?"},{"answer":"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.","question":"What are the advantages of Epic assays compared to traditional (fluorescence, luminescence, radiolabeled) assays?"},{"answer":"Yes. Biochemical assays have successfully been performed with samples in serum and cell lysates. Cell-based assays have successfully been performed in culture media.","question":"Can Epic Technology be used with complex/crude samples?"},{"answer":"Biochemical assays have been run with up to 5 % DMSO and cell-based assays with up to 2.5 % DMSO. In both cell-based and biochemical assays, it is important to match the DMSO concentration of the assay buffer with that of the compounds being tested. A DMSO mismatch will result in a large response that may mask the response of an agonist, in the case of cell based assays, or of a binding event, in the case of biochemical assays. In aggregation assays, an intentional DMSO mismatch is performed.","question":"What is the DMSO tolerance of Epic assays?"}]}]