LUM. SpectraMax Paradigm Dual Color Luminescence (LUM) (BRET2 ) Detection Cartridge. User Guide

Transcription

1 LUM SpectraMax Paradigm Dual Color Luminescence (LUM) (BRET2 ) Detection Cartridge User Guide A September 2010

2 This document is provided to customers who have purchased Molecular Devices, Inc. ( Molecular Devices ) equipment, software, reagents, and consumables to use in the operation of such Molecular Devices equipment, software, reagents, and consumables. This document is copyright protected and any reproduction of this document, in whole or any part, is strictly prohibited, except as Molecular Devices may authorize in writing. Software that may be described in this document is furnished under a license agreement. It is against the law to copy, modify, or distribute the software on any medium, except as specifically allowed in the license agreement. Furthermore, the license agreement may prohibit the software from being disassembled, reverse engineered, or decompiled for any purpose. Portions of this document may make reference to other manufacturers and/or their products, which may contain parts whose names are registered as trademarks and/or function as trademarks of their respective owners. Any such usage is intended only to designate those manufacturers' products as supplied by Molecular Devices for incorporation into its equipment and does not imply any right and/or license to use or permit others to use such manufacturers' and/or their product names as trademarks. Molecular Devices makes no warranties or representations as to the fitness of this equipment for any particular purpose and assumes no responsibility or contingent liability, including indirect or consequential damages, for any use to which the purchaser may put the equipment described herein, or for any adverse circumstances arising therefrom. For research use only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of Molecular Devices, Inc. or their respective owners. These trademarks may not be used in any type of promotion or advertising without the prior written permission of Molecular Devices, Inc. BRET2 is a trademark of PerkinElmer, Inc. Product manufactured by Molecular Devices, Inc Orleans Drive, Sunnyvale, California, United States of America Molecular Devices, Inc. is ISO 9001 registered Molecular Devices, Inc. All rights reserved. Printed in Austria.

3 Safety Information WARNING! If the equipment is used in a manner not specified by Molecular Devices, the protection provided by the equipment may be impaired. Warning and Caution Definitions All Warnings and Cautions in this document include an exclamation point, a lightning bolt, or a light burst symbol framed within a triangle. The exclamation point symbol is an international symbol which serves as a reminder that all safety instructions should be read and understood before installation, use, maintenance, and servicing is attempted. WARNING! A WARNING calls attention to a condition or possible situation that could cause injury to the operator. Electrical Safety CAUTION! A CAUTION calls attention to a condition or possible situation that could damage or destroy the product or the operator s work. When this symbol is displayed in this manual, pay special attention to the specific safety information associated with the symbol. To prevent electrically related injuries and property damage, properly inspect all electrical equipment prior to use and immediately report any electrical deficiencies A 3

4 Safety Information Contact a service engineer for any servicing of equipment requiring the removal of covers or panels. High Voltage WARNING! HIGH VOLTAGE! This symbol indicates the potential of an electrical shock hazard existing from a high voltage source and that all safety instructions should be read and understood before proceeding with the installation, maintenance, and servicing of all modules. Do not remove system covers. To avoid electrical shock, use supplied power cords only and connect to properly grounded (three-holed) wall outlets. Use only multiplug power strips provided by the manufacturer. Disposal of Electronic Equipment It is important to understand and follow all laws regarding the safe and proper disposal of electrical instrumentation. The symbol of a crossed-out wheeled bin on the product is required in accordance with the Waste Electrical and Electronic Equipment (WEEE) Directive of the European Union. The presence of this marking on the product indicates that: the device was put on the European Market after August 13, the device is not to be disposed via the municipal waste collection system of any member state of the European Union. For products under the requirement of WEEE directive, please contact your dealer or local Molecular Devices office for the proper decontamination information and take back program, which will facilitate the proper collection, treatment, recovery, recycling, and safe disposal of the device A

5 (BRET2) Detection Cartridge User Guide Laser Light WARNING! This symbol indicates that a potential hazard to personal safety exists from a laser source. When this symbol is displayed in this manual, pay special attention to the specific safety information associated with the symbol. Laser Specifications The SpectraMax Paradigm Multi-Mode Detection Platform is rated a Class 1 Laser Product because it houses one or more Laser Modules whereas the laser light is not accessible. Figure 2-1 Label on Rear of Instrument A 5

6 Safety Information The embedded Laser Module inside the SpectraMax Paradigm Multi-Mode Detection Platform base instrument is used for the plate height detection and has the following specifications: Laser type: diode laser Wavelength: 650 nm Max. output power: 2 mw, cw Laser Class: Class 2M (IEC , ed. 1.2: 2001) Fan angle: 55 ±5 The embedded laser module is ON, only if the microplate chamber flap is closed (hardware interlock). The user or the service engineer is not exposed to any radiation from the embedded laser module during operation, maintenance, or service. The closed microplate chamber acts as the protective housing. Laser or Laser Diodes Inside the Cartridge Modules Various cartridge modules can have a Laser or Laser Diode (up to Laser Class IV) inside the cartridge modules. The SpectraMax Paradigm Multi-Mode Detection Platform is equipped with a redundant laser safety system. Therefore, the user or the service engineer is not exposed to any radiation of the embedded laser during operation, maintenance, or service A

7 (BRET2) Detection Cartridge User Guide Chemical and Biological Safety Normal operation of the SpectraMax Paradigm Multi- Mode Detection Platform may involve the use of materials that are toxic, flammable, or otherwise biologically harmful. When using such materials, observe the following precautions: Handle infectious samples according to good laboratory procedures and methods to prevent the spread of disease. Observe all cautionary information printed on the original solutions containers prior to their use. Dispose of all waste solutions according to your facility s waste disposal procedures. Operate the SpectraMax Paradigm Multi-Mode Detection Platform in accordance with the instructions outlined in this manual, and take all the necessary precautions when using pathological, toxic, or radioactive materials. Splashing of liquids may occur; therefore, take appropriate safety precautions, such as using safety glasses and wearing protective clothing, when working with potentially hazardous liquids. Use an appropriately contained environment when using hazardous materials. Observe the appropriate cautionary procedures as defined by your safety officer when using flammable solvents in or near a powered-up instrument. Observe the appropriate cautionary procedures as defined by your safety officer when using toxic, pathological, or radioactive materials. Note: Observe all warnings and cautions listed for any external devices attached or used during operation of the Instrument Name. Refer to applicable external device user guides for operating procedures of that device A 7

8 Safety Information Moving Parts Cleaning Maintenance To avoid injury due to moving parts, observe the following: Never attempt to exchange labware, reagents, or tools while the instrument is operating. Never attempt to physically restrict any of the moving components of the SpectraMax Paradigm Multi-Mode Detection Platform. Keep the SpectraMax Paradigm Multi-Mode Detection Platform work area clear to prevent obstruction of the movement. Observe the cleaning procedures outlined in this user guide for the SpectraMax Paradigm Multi-Mode Detection Platform. Prior to cleaning equipment that has been exposed to hazardous material: Appropriate Chemical and Biological Safety personnel should be contacted. The Chemical and Biological Safety information contained in this user guide should be reviewed. Perform only the maintenance described in this manual. Maintenance other than that specified in this manual should be performed only by service engineers. Note: It is your responsibility to decontaminate components of the SpectraMax Paradigm Multi-Mode Detection Platform before requesting service by a service engineer or returning parts to Molecular Devices for repair. Molecular Devices will NOT accept any items which have not been decontaminated where it is appropriate to do so. If any parts are returned, they must be enclosed in a sealed plastic bag stating that the contents are safe to handle and are not contaminated A

9 Contents Safety Information Warning and Caution Definitions Electrical Safety High Voltage Disposal of Electronic Equipment Laser Light Laser Specifications Laser or Laser Diodes Inside the Cartridge Modules Chemical and Biological Safety Moving Parts Cleaning Maintenance SpectraMax Paradigm Dual Color Luminescence (LUM) (BRET2) Detection Cartridge Overview About BRET Luminescence Applications of BRET 2 Luminescence Analyzing BRET 2 Luminescence Data Background Correction Green to Blue Ratios Detection Limit Sample Volumes and Concentration of Reactants Data Optimization Performing Measurements Microplate Recommendations Measurement Specifications A 9

10 Contents Preventive Maintenance and Troubleshooting Validating Performance with the Multi-Mode Validation Plate Troubleshooting A

11 SpectraMax Paradigm Dual Color Luminescence (LUM) (BRET2) Detection Cartridge Overview The SpectraMax Paradigm Dual Color Luminescence (LUM) (BRET2 ) Detection Cartridge is for use with the SpectraMax Paradigm Multi-Mode Detection Platform. Installing this cartridge enables detection methods for glow luminescence and PerkinElmer BRET 2 reagents. 1 Note: For glow luminescence measurements that do not require emission filters, the Glow Luminescence (LUM) Detection Cartridges (PN , PN , or PN ) optimized for various plate formats should be used. A luminescence detection cartridge contains the optics and electrical components needed to perform specific measurement modes or for specific applications. The SpectraMax Paradigm Multi-Mode Detection Platform requires these detection cartridges to enable different measurement modes. The SpectraMax Paradigm Multi-Mode Detection Platform is a modular multi-mode microplate detector. Userinstallable and removable detection cartridges allow the detector to be configured for specific applications and easily expand the capabilities of the detector at any time. 1. BRET 2 is a trademark of PerkinElmer, Inc., Waltham, Massachusetts, USA A 11

12 (BRET2) Detection Cartridge The SpectraMax Paradigm Dual Color Luminescence (LUM) (BRET2) Detection Cartridge occupies one (1) slot. It may be installed in either the top read detection cartridge transport for top reading, or in the bottom read detection cartridge transport for bottom reading. However, it may NOT be installed in the rearmost slot on either the top or bottom detection cartridge transport. Refer to Installing Detection Cartridges in the SpectraMax Paradigm Multi-Mode Detection Platform User Guide, for installation instructions. Note: It is recommended to install the SpectraMax Paradigm Dual Color Luminescence (LUM) (BRET2) Detection Cartridge in the top read detection cartridge transport when running glow luminescence detection methods. About BRET Luminescence Bioluminescence Resonance Energy Transfer (BRET) is a measurement technique sharing the key feature of Fluorescence RET (FRET) while using the advantages of luminescence reading. BRET employs a luminescence substrate that may undergo cleavage due to an enzymatic group (donor label) resulting in luminescence emission (channel 1). Unlike other, generic luminescence assays of such kind, the BRET luminescence may be quenched by a radiationless Energy Transfer to an acceptor label (a fluorophore) resulting in fluorescence emission at a longer wavelength (red shifted, channel 2). The acceptor must be chosen such that its excitation spectrum overlaps the emission spectrum of the luminescence (Resonance condition). In the presence of substrate the energy transfer can only occur when the enzymatic group and the acceptor are in close proximity. Because of this fact, when labels are applied to study protein-protein interactions for example, signal measured at the emission wavelength of the acceptor fluorophore indicates binding A

13 (BRET2) Detection Cartridge User Guide The SpectraMax Paradigm Dual Color Luminescence (LUM) (BRET2) Detection Cartridge contains the components for measuring the light intensity from BRET 2 reagents. Since the light is emitted as a result of a chemical reaction, no excitation light and no excitation filters are required to measure luminescence, and the detection of the acceptor fluorophore (channel 2) can be regarded as a luminescence also. Emission filters differentiate emissions from both the luminescence and the acceptor fluorophore. Thus, a dual color luminescence method is applied. The SpectraMax Paradigm Multi-Mode Detection Platform incorporates a dual emission design to measure both emission channels simultaneously, improving precision and reducing read times. CAUTION! Luminescence light levels visible to the human eye may cause damage to the detection system. Applications of BRET 2 Luminescence BRET 2 can be used to study various kinds of proteinprotein interactions. It employs a luciferase label (where label typically means fused by the use of expression vectors) for the one protein and a Green Fluorescent Protein (GFP) variant label for the other. The type of luciferase (Renilla) metabolizes the luminescent substrate (a coelenterazine derivative) resulting in blue luminescence (emission filter 1). Given the substrate, when the other protein with the GFP label is in close proximity to the luciferase, it quenches the blue luminescence and emits a green luminescence (emission filter 2). Thereby, a high green to blue ratio indicates binding. The energy transfer is possible by the selection of the labels, that is, the overlap of the blue luminescence spectrum with the excitation spectrum of the GFP (resonance condition). As a result, BRET 2 combines the advantage of being a homogeneous (no washing steps required) assay with the generic sensitivity of luminescence detection. See Green to Blue Ratios on page A 13

14 (BRET2) Detection Cartridge The BRET 2 reagents can be applied in various ways to study other types of molecular interactions in cell based assays. For typical applications, refer to the manufacturer s website at Analyzing BRET 2 Luminescence Data The conversion rate of photons to counts is individual for each detector. Therefore, raw data from the same plate may appear significantly different from one instrument to the next. In addition, the data format used by other manufacturers may not be counts per second and may be different by several orders of magnitude. It is important to know that the number of counts and the size of figures is in no way an indication of sensitivity. See Detection Limit on page 16. On the SpectraMax Paradigm Multi-Mode Detection Platform, raw data is normalized to counts per second by dividing the number of counts by the read time per well. Analyzing and validating dual color luminescence intensity data from BRET 2 reagents typically consists of the following: Background Correction on page 15 Green to Blue Ratios on page 15 Detection Limit on page 16 Sample Volumes and Concentration of Reactants on page 16 Data Optimization on page A

15 (BRET2) Detection Cartridge User Guide Background Correction The light detected in a luminescent measurement typically has two components: specific light from the luminescent reaction; and an approximately constant level of background light caused by various factors, including the plate material and impurities in the reagents. The background can be effectively measured using blank replicates. Blanks should include the luminescent substrate (chemical energy source) but not the luminescence agent (typically an enzymatic group which makes the substrate glow). For improved results, run replicates for all blanks, controls, and samples. In this case, the blank value subtracted is the average value of all blanks. Multi-Mode Analysis Software performs automatic blank averaging and reduction if blanks are defined on the plate layout in the protocol. Refer to Creating Protocols in the Multi- Mode Analysis Software User Guide. Green to Blue Ratios Energy Transfer data are typically normalized by taking the red shifted emission and dividing it by the blue shifted emission. In terms of the BRET 2 reagents, the normalization of the green signal with the blue signal mainly compensates for the decay of the signal due to the digestion of the substrate. The Multi-Mode Analysis Software supports data reduction in taking ratios of both emission channels. Because of the signal decay, measure the assay immediately after adding the luminescence substrate. The spectral separation of the green from the blue signal conveniently eliminates the need for preprocessing the raw data A 15

16 (BRET2) Detection Cartridge Detection Limit The detection limit is the smallest measurable signal above the background. The background can be determined as described above, taking a number of blank measurements and calculating an average value and standard deviation for the blanks. The detection limit can then be determined as the average blank plus three standard deviations. If the average sample value measures above the detection limit, the sample can be detected at a statistically significant level. The detection limit can be described by the following equation: det limit = 3 x stdev(blank) / (standard-blank) x conc std (equation 1) where standard is the signal of a standard, blank is the mean of blanks, conc std is the concentration of the standard, and stdev(blank) is the standard deviation of the blanks. Sample Volumes and Concentration of Reactants The concentration of the luminescent agent impacts the amount of light output in a luminescent reaction. Light is emitted as a result of a reaction between two or more compounds; therefore, the amount of light output is proportional to the amount of the limiting reagent in the sample. For example, in a luciferase assay system, when total volume is held constant and luciferase is the limiting reagent, the blanked light output is proportional to the concentration of luciferase in the sample. At very high concentrations of luciferase, the excess requirement for the availability of substrate may become violated because considerable amounts of substrate are used up, resulting in earlier decay of the strongest samples. In effect, it may appear to be a non-linearity of the detector, while it actually is an effect of the assay. Note: Very bright samples can exceed the linear dynamic range of the detection system. In such case, reading can be performed using an attenuation filter A

17 (BRET2) Detection Cartridge User Guide Data Optimization The measurement noise is dependent on the read time per sample (time per plate or time per well). In particular, the detection limit improves when the read time is increased. Therefore, it is important to specify the read time when comparing measurements. All low light level detection devices have some measurement noise in common. In order to average out the measurement noise, optimization of the time per plate (or time per well) shall target for accumulating as many counts as possible. Within some range, the reduction of noise (CVs, detection limit) can be accomplished by increasing the read time per well, as far as is acceptable from throughput and sample stability considerations. For maximum sensitivity, the BRET 2 reagents in particular shall be read immediately after the last preparation step adding the luminescence substrate. When performing a binding assay, controls are involved that represent a maximum (high) and a minimum (low) G/B (green to blue) ratio, and the Z' parameter may be applied. Beyond merely taking the ratio of high and low G/B values, the Z' includes replicate variability to validate the actual data quality. Note: Z is a qualification parameter that can be used to validate the results of a measurement. It is a function of assay resolution (equation 2), which is a ratio of the average standard deviation of high and low control replicates (S) to the assay window (W) the difference between the average value of the high controls and the average value of the low controls. Z'=1 6 S W ---- (equation 2) A 17

18 (BRET2) Detection Cartridge Performing Measurements When the SpectraMax Paradigm Dual Color Luminescence (LUM) (BRET2) Detection Cartridge is installed in the SpectraMax Paradigm Multi-Mode Detection Platform, detection methods and protocols to perform glow luminescence measurements are enabled in Multi-Mode Analysis Software. The SpectraMax Paradigm Dual Color Luminescence (LUM) (BRET2) Detection Cartridge enables a protocol template in Multi-Mode Analysis Software that may be used to create protocols. For details see the template info. Additional detection methods and protocols using the (BRET2) Detection Cartridge may be created using Multi- Mode Analysis Software. For information on creating detection methods using the SpectraMax Paradigm Dual Color Luminescence (LUM) (BRET2) Detection Cartridge, refer to Creating and Editing Detection Methods in the Multi-Mode Analysis Software User Guide. For information on creating and running protocols using the SpectraMax Paradigm Dual Color Luminescence (LUM) (BRET2) Detection Cartridge, refer to Creating and Running Protocols in the Multi-Mode Analysis Software User Guide. Note: Measurement protocols that use multiple cartridges may also be created and run on the SpectraMax Paradigm Multi-Mode Detection Platform A

19 (BRET2) Detection Cartridge User Guide Microplate Recommendations The type of microplate and the way it is handled can affect the measurement performance of the instrument. Plate height may not exceed 25 mm. Select a plate type with properties suited for the application and for use with multi-mode detectors. Table 1-1 lists microplate selection guidelines. Note: For each type of assay (defined by microplate type and sample volume), it is recommended to optimize the labware. Because the luminescence cartridge features a read head that moves towards the microplate into close proximity, the actual read height of the labware must be defined accurately. Refer to Creating and Editing Labware in the Multi-Mode Analysis Software User Guide. Plate handling guidelines include: Keep unused plates clean and dry. Make sure the strips on strip plates are inserted correctly and level with the frame. Table 1-1 Microplate Selection Guidelines Measurement Technique Dual Color Luminescence Microplate Type solid white Additional Considerations When an application specifies a surface treatment, only use plates with the correct treatment. Note: White plates provide significantly higher signal than black plates, and are recommended if high sensitivity is required. White plates may, however, exhibit some detectable phosphorescence which increases background after being exposed to light (in particular under neon lights). For maximum sensitivity, it is recommended to prepare plates under reduced ambient light conditions A 19

20 (BRET2) Detection Cartridge Measurement Specifications The specifications for measurements using the (BRET2) Detection Cartridge are shown in Table 1-2. Table 1-2 Measurement Specifications Parameter Detection Cartridge Name Specification Short Name Part Number Weight 1.5 lbs (0.7 kg) SpectraMax Paradigm Dual Color Luminescence (LUM) (BRET2) Detection Cartridge LUM-BRET2 Detection Dual Color Luminescence Techniques Type Dual emission Number of Slots 1 Top/Bottom Top or Bottom, except for rear-most slot Light Source NA Wavelength Range Labels/Substrates EM1: EM2: Any compatible with wavelength range Applications Designed for use with PerkinElmer BRET 2 reagents Validation Plate test names ending with -BRET2 Tests A

21 (BRET2) Detection Cartridge User Guide Preventive Maintenance and Troubleshooting To ensure optimum operation of the instrument, perform the following preventive maintenance procedures as necessary: Open the detection cartridge transport only when removing or installing detection cartridges. When the detection cartridge is not in use, always keep the red cap in place and store in the detection cartridge box. Follow appropriate decontamination procedures as instructed by the laboratory safety officer. Contact a Molecular Devices service engineer to inspect the instrument every two years. Use a Multi-Mode Validation Plate to regularly validate the performance of the cartridge. Contact Molecular Devices sales or service for more details. Respond appropriately to any error messages displayed by Multi-Mode Analysis Software. Validating Performance with the Multi-Mode Validation Plate The Multi-Mode Validation Plate is used to validate the performance of absorbance, fluorescence, and luminescence measurements performed on the SpectraMax Paradigm Multi-Mode Detection Platform. Validation runs performed with the validation plate only validate detection cartridge performance; factory- and user-defined settings are not changed. The tests that need to be performed to validate performance differ between detection cartridges. To validate the measurement performance of the (BRET2) Detection Cartridge, run all validation plate test suites with the detection cartridge short name LUM-BRET2 appended to the end of the test name A 21

22 (BRET2) Detection Cartridge Note: Refer to the Multi-Mode Validation Plate User Guide for information on running tests with the validation plate. Troubleshooting WARNING! Only officially trained service engineers may perform service procedures on the instrument. Contact a Molecular Devices service engineer when service is required. Perform the following troubleshooting techniques when necessary. Also refer to Performing Basic Maintenance and Troubleshooting in of the SpectraMax Paradigm Multi-Mode Detection Platform User Guide. Table 1-3 Troubleshooting the SpectraMax Paradigm Multi-Mode Detection Platform If Functional failure Detection cartridge fails validation tests with validation plate Then Install the cartridge in a different detection cartridge slot. Contact Molecular Devices Technical Support. Refer to the Multi-Mode Validation Plate User Guide. Contact Molecular Devices Technical Support A