Amersham - GE Healthcare Life Sciences
Amersham
SPA product guide
Amersham SPA product guide Contents Scintillation proximity assay—versatile technology for rapid and sensitive assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 The principle of SPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Advantages of SPA bead-based assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Choosing between SPA Scintillation Beads and SPA Imaging Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 SPA bead types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 SPA bead coatings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 Suitable radioisotopes for SPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 Application of SPA technology to enzyme assays
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Assay format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 SPA bead types and coatings used for enzyme assays . . . . . . . . . . . . . . . . . . . .8 Application of SPA technology to kinase assays
. . . . . . . . . . . . . . . . . . . . . . . . . .9
SPA bead types and coatings used for kinase assays . . . . . . . . . . . . . . . . . . . . . .9 Application of SPA technology to receptor-binding assays . . . . . . . . . . .11 SPA bead types and coatings for receptor-binding assays . . . . . . . . . . . . . .12 Typical applications for receptor-binding SPAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 Application of SPA technology to molecular interaction research . .14 SPA bead types and coatings for studying molecular interactions
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Application of SPA technology to radioimmunoassays . . . . . . . . . . . . . . . .16 Application of SPA technology to cell-based assays . . . . . . . . . . . . . . . . . . . .17 Principle of Cytostar-T Scintillating Microplates
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Application of SPA technology to drug metabolism and pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 Receptor Membrane Preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 Scintillation counter settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 Multiwell plates suitable for counting SPA Scintillation Beads . . . . . . .20 Multiwell plates suitable for measuring SPA Imaging Beads
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Ordering information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22 Related products
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Scintillation proximity assay—versatile technology for rapid and sensitive assays Scintillation proximity assay (SPA) is a homogeneous and versatile technology for the rapid and sensitive assay of a wide range of biological processes. The assay format requires no separation steps, is amenable to automation, and is supported by more than 10 years of application data generated independently and by GE Healthcare scientists. SPAs using either SPA Scintillation Beads or SPA Imaging Beads can be read on scintillation counters and CCD imagers respectively.
Versatile A range of bead types and coatings • For use with enzyme and receptor targets, radioimmunoassays, and molecular interactions • Various pack sizes available from stock for assay development and research • Bulk packs available for screening applications • Custom bead coatings on request SPA beads are suitable for use with a range of radioisotopes • 3H, 125I, 33P, and 14C can be used as radiolabels • No requirement for protein modification Compatible with any scintillation counter or CCD imager • SPA Scintillation Beads can be used in tubes and 96- or 384-well microplates • SPA Imaging Beads can be used in any format microplate
No separation step Reduction in handling steps • Increased assay precision and assay reproducibility • Improved safety • No need for filters and scintillation cocktails Reduction in radioactive liquid waste • Decreased disposal costs Amenable to automation • Increased throughput
Proven track record • Leading radiometric screening technology • Supported by more than 10 years of application data generated by GE Healthcare and leading pharmaceutical companies • Over 600 literature references are available on our Web sites at www.gehealthcare.com/spa and www.gehealthcare.com/leadseeker.
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The principle of SPA When certain radioactive atoms decay, they release ß-particles. The distance these particles travel through an aqueous solution is dependent on the energy of the particle. If a radioactive molecule is held in close enough proximity to a SPA Scintillation Bead or a SPA Imaging Bead, the decay particles stimulate the scintillant within the bead to emit light, which is then detected in a PMT-based scintillation counter or on a CCD-based imager respectively (Table 1). However, if a radioactive molecule is free in a solution containing SPA beads, the decay particles will not have sufficient energy to reach the bead and no light will be emitted. This discrimination of binding by proximity means that no separation of bound and free ligand is required.
Free radiolabel
Bound radiolabel
Fig 1. SPA principle.
Advantages of SPA bead-based assays SPA bead-based assays have many advantages over plate-based methods • The higher surface area of SPA beads allows the concentration of key assay components (e.g. receptor membrane) to be increased or decreased, thus giving greater flexibility in assay design. • The higher binding capacity of SPA beads gives higher signal allowing a potential reduction in the amount of radioactive label required. • The amount of SPA bead can be altered to optimize the sensitivity of an assay. • SPA beads are format independent making it easier to transfer assays to higher density plates. • A choice of four different core bead matrices allows optimization of signal and background over a wide range of applications (Table 2). • SPA beads are not dependent on one plate type or supplier (Table 11).
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Choosing between SPA Scintillation Beads and SPA Imaging Beads The choice of SPA bead depends on several parameters and these are summarized in Table 1. Table 1. SPA bead selection guide
SPA Scintillation SPA Imaging Beads Beads
Feature
Benefit
PMT readers, standard beta counters
CCD Imagers
SPA Scintillation Beads emit in the blue region and SPA Imaging Beads emit light in the red region of spectra (Fig 2).
Match bead to instrument type (e.g. CCD imagers are more sensitive in the red region).
Throughput Medium to high
High to ultra high
Choice of throughput
Match instrument and bead choice with throughput needs
Format
96- or 384well plates
96-, 384-, 1536-well Choice of format plates and above
Increase throughput and decrease assay component costs
Emission
Blue region (400 nm)
Red region (615 nm)
Reduce color quench caused by orange/red colored compounds through use of SPA Imaging Beads
Instrument
Choice of emission properties
Different colored dyes can affect the signal produced in a SPA using either SPA Scintillation Beads or SPA Imaging Beads (Fig 3). The dyes used to generate the data in Figure 3 were at concentrations of up to 3 mg/ml, which is in excess of that liable to be found in typical compound libraries, while all wells have identical amounts of radiolabel and SPA bead. The data show no significant attenuation of signal from the SPA Imaging Beads at any concentration of tartrazine, whereas the SPA Scintillation Beads report a lower level of signal as dye concentration increases. The presence of Chicago Sky Blue (CSB) causes quenching of the SPA Imaging Bead signal from a concentration of 0.03 mg/ml; the YSi and PVT beads only begin to show quenching at 0.1 mg/ml of CSB. Given that there is a higher proportion of yellow/orange/red compounds than blue compounds in most compound libraries, the issue of color quenching is reduced by conversion to SPA Imaging Beads.
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Relative intensity
Fig 2. Wavelength emissions from SPA Scintillation Beads (PVT and YSi) and SPA Imaging Beads (PS and YOx ). The overlaid spectrum indicates the compound colors that have the potential to quench bead signal.
Wavelength (nM)
Tartrazine
Fig 3. Plate map showing effect of color quenching on an imaged assay, on SPA Scintillation Beads (PVT and YSi), and SPA Imaging Beads (PS and YOx).
Chicago Sky Blue
SPA bead types SPA Scintillation Beads and SPA Imaging Beads are available in different core materials and their benefits are described in Table 2. Table 2. SPA bead type selection guide.
SPA Scintillation Beads SPA Imaging Beads
Benefit
Polyvinyltoluene (PVT)
Polystyrene (PS)
Easy to dispense and suited to automation and high-throughput screening. Used for the majority of SPA scintillation and SPA imaging applications
Yttrium silicate (YSi)
Yttrium oxide (YOx)
Possess greater scintillation counting efficiency but less suited to automation because their higher density makes them settle more rapidly than PVT or PS beads
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SPA bead coatings SPA beads are coated with coupling molecules such as wheat germ agglutinin (WGA), streptavidin, antibodies, glutathione, and copper or nickel chelate. These coupling molecules allow a variety of assay reagents such as cell membranes, biotinylated peptides, and fusion proteins to be captured onto the bead. The choice of bead coating will be determined by the application under investigation. Table 3 gives guidance on which bead coating is most suited to a range of typical applications. Table 3. SPA bead coatings and their recommended applications.
Bead coating
Bead types available
Application
Arginine Binding
YSi
Capture of arginine
Charge-Based Binding
YOx
Capture of GST fusion substrates
Copper chelate (His-Tag) Nickel Chelate (His-Tag) Glutathione Streptavidin
PVT,YSi PS, YOx YSi PVT, YSi, PS, YOx,
Enzyme assays, molecular interactions
Wheat Germ Agglutinin (WGA) Membrane Binding Polyethyleneimine (PEI) Polylysine Select-a-Bead Kit*
PVT, YSi, PS, YOx YOx PS, PVT PS, YSi PVT, YSi, PS, YOx
Receptor-binding
Anti-Mouse Anti-Rabbit Anti-Sheep Protein A
PVT, YSi, PS, YOx PVT, YSi, PS, YOx PVT, YSi PVT, YSi, PS, YOx
RIA, enzyme assays, molecular interactions
PDE
YSi
PDE activity
Suitable radioisotopes for SPA The most important characteristic when selecting a radioisotope for SPA is the pathlength of the decay particle (Table 4). In general, the shorter the pathlength of the decay particle, the more suited it is to SPA. • Tritium and iodine-125 are ideally suited to SPA • Carbon-14, sulfur-35, and phosphorus-33 have been used successfully with SPA Table 4. Radioisotopes suitable for SPA and their decay particle average pathlengths.
Isotope
Average pathlength of decay particle in aqueous solution
Tritium
1.5 μm
Iodine-125
2e-: 1.0 μm, 17 μm
Carbon-14
50 μm
Sulfur-35
65 μm
Phosphorus-33
125 μm
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Application of SPA technology to enzyme assays Enzyme SPA applications can be used to identify potential new enzyme inhibitors, measure enzyme activity, and perform kinetic analysis. Activity of the following enzymes can be determined by enzyme SPA . • Hydrolases such as proteases and nucleases • DNA modifying enzymes such as helicase and integrase • Transferases • Phosphodiesterases • Lipid modifying enzymes Examples of enzyme SPA applications can be found as Proximity News articles on our SPA Web site at www.gehealthcare.com/spa and as Leads articles on our LEADseeker™ Web site at www.gehealthcare.com/leadseeker. These Web sites also include bibliographies containing further publications citing enzyme SPA applications.
Assay format The format of an enzyme SPA depends on the action of the enzyme being studied. 1. Signal increase assays are used to measure the activity of kinases, polymerases, and transferases because these enzymes add radiolabeled molecules to the substrate immobilized on the bead (Fig 4a). 2. Signal decrease assays are used to measure the activity of enzymes such as proteases and nucleases because they cleave radiolabeled substrate from the bead (Fig 4b).
a.
b.
Fig 4. Enzyme SPA concept. (a) Signal increase format (b) Signal decrease format.
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SPA bead types and coatings used for enzyme assays 1. The majority of enzyme SPAs use biotinylated substrates that may be either immobilized on, or subsequently captured by, streptavidin-coated SPA Scintillation Beads or SPA Imaging Beads. The biotin-streptavidin system provides a reliable and reproducible high-affinity capture mechanism. Further details on biotinylation techniques can be found in Proximity News 39, a copy of which is available on our SPA Web site at www.gehealthcare.com/spa. 2. Arginine Binding YSi SPA Beads are used in the Nitric Oxide Synthase (NOS) Screening Kit (TRKQ7160). Figure 5 shows timecourse data whereby [3H]arginine is converted to citrulline by the action of NOS. The product, [3H]citrulline, does not bind to the beads and the action of NOS is therefore observed as a reduction in SPA counts.
Fig 5. Timecourse data for the Nitric Oxide Synthase (NOS) Screening Kit.
3. GST-tagged fusion protein enzyme substrates can be captured onto SPA beads coated with glutathione. 4. His-tagged fusion proteins can be captured onto copper chelate coated SPA Scintillation Beads, nickel chelate coated SPA imaging Beads or charge based binding SPA Imaging Beads. Table 5. SPA bead types and coatings commonly used in enzyme SPA applications.
Instrument
Core bead type
Coating
Product code
PMT readers
PVT
Streptavidin Copper chelate
RPNQ0006, RPNQ0007, RPNQ0009 RPNQ0095
YSi
Streptavidin Copper chelate Glutathione PDE Arginine binding SPA beads
RPNQ0012, RPNQ0015 RPNQ0096 RPNQ0033, RPNQ0034 RPNQ0150 RPNQ0101
PS
Streptavidin Nickel chelate
RPNQ0261, RPNQ0263 RPNQ0266, RPNQ0267
YOx
Streptavidin Nickel chelate Charge-based binding
RPNQ0271, RPNQ0273 RPNQ0276, RPNQ0277 RPNQ0320
CCD imagers
Bulk order quantities are available for larger scale research experiments and high-throughput screens.
For a range of fully optimized enzyme SPA kits, please visit our Web site at www.gehealthcare.com/spa. A full listing of SPA beads is shown on page 23. 8
Application of SPA to kinase assays Kinases play pivotal roles in many signal transduction cascades and consequently kinase activities remain a key focus of academic and pharmaceutical research. GE Healthcare has developed kinase SPA applications for tyrosine and serine/threonine kinases using both peptide and protein substrates. • Cyclin dependent kinases
• MAP kinases
• Protein kinase A
• Protein kinase C isoforms
The basic principle of kinase SPA involves the transfer of [33P]phosphate from [33P]ATP to the tyrosine, serine, or threonine in the peptide or protein substrate. The phosphorylated peptide is then captured by a streptavidin-coated SPA bead (Fig 6).
Kinase enzyme
Biotinylated peptide or MBP [33P]-ATP substrate
Streptavidin SPA Imaging Beads
ADP
Fig 6. Kinase SPA concept.
SPA bead types and coatings used for kinase assays Typically, kinase SPAs are configured by using streptavidin-coated SPA beads and biotinylating the peptide or protein substrate. Alternatively, fusion protein binding SPA beads can also be used. A list of kinase applications can be found in the bibliography section on our Web sites at www.gehealthcare.com/spa and www.gehealthcare.com/leadseeker.
Fig 7. Inhibition of p56lck kinase activity by PP2 and nocodazole in 1536-well format using Polystyrene SPA Imaging Beads. Each data point is the mean ± SEM of six replicates.
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Table 6. SPA bead types and coatings commonly used in kinase SPA applications.
Instrument
Core bead type
Coating
Product code
PMT readers
PVT
Streptavidin Copper chelate
RPNQ0006, RPNQ0007, RPNQ0009 RPNQ0095
YSi
Streptavidin Copper chelate
RPNQ0012, RPNQ0015 RPNQ0096
PS
Streptavidin Nickel chelate
RPNQ0261, RPNQ0263 RPNQ0266, RPNQ0267
YOx
Streptavidin Nickel chelate Charge-based binding
RPNQ0271, RPNQ0273 RPNQ0276, RPNQ0277 RPNQ0320
CCD imagers
Bulk order quantities are available for larger scale research experiments and high-throughput screens.
For a range of fully optimized enzyme SPA kits, please visit our Web site at www.gehealthcare.com/spa. A full listing of SPA beads is shown on page 23.
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Application of SPA technology to receptor-binding assays Receptor-binding SPAs can be configured to determine receptor kinetics, saturation binding, or to detect inhibitors of radioligand binding. SPA has been successfully applied to receptor-binding assays by immobilizing receptors directly to SPA beads via a number of coupling methods. SPA receptor-binding assays have been developed for a wide range of receptor types, including: • Serotonin • Acetylcholine • Chemokine • Cytokine • Steroid • Inositol triphosphate SPA technology also enables the binding of GTP to G-protein coupled receptors (GPCRs) following agonist binding to the GPCR to be measured. SPA is compatible with receptors prepared from a number of different sources: • Cell membrane preparations from tissue • Cell membrane preparations from cultured cells • Soluble purified receptors • Solubilized receptors from tissues and cultured cells The principle of receptor-binding SPAs involves radiolabeled ligand binding to a receptor immobilized on the surface of a SPA bead. The bound ligand is held in close enough proximity to the bead to stimulate scintillant within the bead to emit light. Unbound radioligand is too distant from the bead to transfer energy and therefore goes undetected (Fig 8).
Bead emitting light Fig 8. Receptor-binding SPA principle.
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SPA bead types and coatings used for receptor-binding assays 1. Receptor-binding SPAs are usually formatted using wheat germ agglutinin (WGA)-coated SPA beads. WGA binds N-acetyl-ß-D-glucosaminyl residues, N-acetyl-ß-D-glucosamine oligomers, and glycoproteins present in cell membranes to capture cell membranes expressing the receptor of interest. 2. The use of polyethyleneimine to either treat WGA or as a separate bead coating can further reduce non-specific binding, 3. Poly-L-lysine is used for binding negatively charged cellular membranes. 4. Soluble and solubilized receptors can be captured onto antibody binding SPA beads via an antibody specific to the receptor. 5. The receptor protein can be biotinylated and captured onto streptavidin-coated beads.
Typical applications for receptor-binding SPAs Proximity News, 27, describes the steps required to develop a receptor-binding SPA and can be found on our SPA Web sites at www.gehealthcare.com/spa and www.gehealthcare.com/leadseeker. Further examples of receptor-binding SPA applications can be found as Proximity News articles on the SPA Web site, which also includes a bibliography containing over 170 publications citing receptor-binding SPA applications. Application notes detailing receptor-binding applications using SPA Imaging Beads can be found at www.gehealthcare.com/spa. Data from a typical receptor-binding assay is shown in Figure 9 whereby competition analysis was performed on membranes from HEK cells expressing the human melanocortin 4 receptor. The radiolabeled ligand, [125I]NDP-α-MSH - [Nle4-D-Phe] melanocyte stimulating hormone and the competitor (unlabelled NDP-α-MSH) were added together with WGA-coated Polystyrene SPA Imaging Beads at T = 0 into the wells of 384-well plates. The assays were incubated overnight at room temperature without shaking and the plates were then read for 5 min on the LEADseeker Multimodality Imaging System.
Fig 9. Melanocortin 4 receptor imaging SPA competition analysis. Values shown as means +/- SD (n = 3).
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Table 7. SPA bead types and coatings that may be used in receptor-binding assays.
Instrument
Core bead type Coating
Product code
PMT readers
PVT
RPNQ0001, RPNQ0252 RPNQ0003 RPNQ0004 RPNQ0097 RPNQ0019, RPNQ0031, RPNQ0069 RPNQ0011 RPNQ0010 RPN143, RPNQ0068 RPNQ0250 RPNQ0260, RPNQ0262 RPNQ0286, RPNQ0287 RPNQ0288, RPNQ0289 RPNQ0098 RPNQ0264, RPNQ0265 RPNQ0294, RPNQ0295 RPNQ0274, RPNQ0275 RPNQ0291
YSi
CCD imagers
PVT and YSi PS
YOx PS and YOx
WGA WGA PEI Type A WGA PEI Type B Polyethyleneimine (PEI) Protein A WGA Poly-L-lysine Protein A SPA Select-a-Bead Kit* WGA WGA PEI Type A WGA PEI Type B PEI Protein A Poly-L-lysine Protein A SPA Imaging Select-a–Bead Kit†
*Contains 100 mg each of WGA PVT; WGA YSi; WGA PEI Type A, WGA PEI Type B, and poly-L-lysine to allow quick and convenient receptor assay development using SPA Scintillation Beads. †Contains 50 mg each of WGA PS; WGA YOx; WGA PEI Type A, WGA PEI Type B, and poly-L-lysine to allow quick and convenient receptor assay development using SPA Imaging Beads.
Bulk order quantities are available for larger scale research experiments and high-throughput screens. A range of fully optimized SPA kits for GPCR ligand binding are also available (see Ordering information) or visit our Web sites at www.gehealthcare.com/spa and www.gehealthcare.com/leadseeker. A full listing of SPA beads is shown on page 23.
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Ordering information Product
Quantity
Code number
Adrenergic a2a [3H]SPA GPCR Assay
Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays
MRP0150-250UCI MRP0151-250UCI MRP0010-250UCI MRP0011-250UCI MRP0020-10UCI MRP0021-10UCI MRP0030-10UCI MRP0031-10UCI MRP0040-10UCI MRP0041-10UCI MRP0050-10UCI MRP0051-10UCI MRP0060-250UCI MRP0061-250UCI MRP0170-250UCI MRP0171-250UCI MRP0070-10UCI MRP0071-25UCI MRP0080-10UCI MRP0081-10UCI MRP0090-250UCI MRP0091-250UCI MRP0110-10UCI MRP0111-20UCI MRP0100-10UCI MRP0101-10UCI MRP0120-10UCI MRP0121-10UCI MRP0130-250UCI MRP0131-250UCI MRP0140-10UCI MRP0141-10UCI
Adrenergic a2c [3H]SPA GPCR Assay CCR6 [125I]SPA GPCR Assay CGRP [125I]SPA GPCE Assay CXCR2 [125I]SPA GPCR Assay CXCR3 [125I]SPA GPCR Assay Histamine H1 [3H]SPA GPCR Assay Histamine H3 [3H]SPA GPCR Assay Melanocortin MC4 [125I]SPA GPCR Assay Melanocortin MC5 [125I]SPA GPCR Assay Muscarinic M1 [3H]SPA GPCR Assay Neurokinin 2 [125I]SPA GPCR Assay Neuromedin NMU-1R [125I]SPA GPCR Assay Nociceptin ORL-1 [125I]SPA GPCR Assay Serotonin 5HT2c [3H]SPA GPCR Assay Somatostatin sst4 [125I]SPA GPCR Assay
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Application of SPA technology to molecular interaction research SPA technology has been successfully applied to the study of a variety of molecular interactions. • Protein:protein interactions
• Protein:peptide interactions
• Protein:DNA interactions
• Cell adhesion molecule interactions
Many low-affinity binding events, such as occur between cell adhesion molecules, can be disrupted by the separation and wash steps required in heterogeneous techniques such as filtration. However, with SPA, these low-affinity binding events can be studied because there is no separation step.
SPA bead types and coatings for studying molecular interactions Various SPA bead types and coatings have been used for molecular interaction applications (Table 8): 1. Biotinylated peptides derived from SH2 binding proteins have been captured onto streptavidin-coated PVT SPA Scintillation Beads enabling the binding of a radiolabeled SH2 protein to the peptide to be observed. 2. Antibody capture assay formats utilizing antibody-binding SPA beads have been used to follow the binding of radiolabeled proteins, peptides, or in the case of transcription factors, DNA. 3. Glutathione and copper and nickel chelate coated fusion protein SPA Beads offer a more direct coupling method that does not require either additional reagents such as antibodies, or protein modification such as biotinylation. 4. Charge-based binding YOx SPA Imaging beads enable the capture and assay of glutathione S-transferase (GST) fusion proteins. The type of assay format feasible with the fusion protein binding SPA beads is shown in Figure 10.
Fig 10. Typical assay formats for molecular interaction SPA using glutathione or copper chelate coated SPA beads. 15
Table 8. SPA bead types and coatings commonly used in molecular interaction SPA applications.
Instrument
Core bead type
Coating
Product code
PMT readers
PVT
Protein A Anti-rabbit antibody Anti-mouse antibody Anti-sheep antibody Streptavidin Copper chelate
RPNQ0019 RPNQ0016 RPNQ0017 RPNQ0018 RPNQ0006, RPNQ0007, RPNQ0009 RPNQ0095
YSi
Protein A antibody Anti-Rabbit antibody Anti-mouse antibody Anti-sheep antibody Streptavidin Copper chelate Glutathione
RPN143 RPN140 RPN141 RPN142 RPNQ0012, RPNQ0015 RPNQ0096 RPNQ0033, RPNQ0034
PS
Streptavidin Nickel chelate
RPNQ0261, RPNQ0263 RPNQ0266, RPNQ0267
YOx
Streptavidin Nickel chelate Protein A Charge based binding
RPNQ0271, RPNQ0273 RPNQ0276, RPNQ0277 RPNQ0274, RPNQ0275 RPNQ0320
CCD imagers
A full listing of SPA beads is shown on page 23.
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Application of SPA technology to radioimmunoassays Scintillation proximity radioimmunoassay is based on the reaction of antibody-bound ligand with SPA beads coated with either a secondary antibody or protein A. The range of SPA beads designed for use in RIA allows the conversion of heterogeneous RIA systems into the homogeneous SPA format. Table 9. SPA bead types and coatings that may be used in SPA radioimmunoassays.
Instrument
Core bead type
Coating
Product code
PMT readers
PVT
Protein A Anti-rabbit antibody Anti-mouse antibody Anti-sheep antibody
RPNQ0019 RPNQ0016 RPNQ0017 RPNQ0018
YSi
Protein A antibody Anti-rabbit antibody Anti-mouse antibody Anti-sheep antibody
RPN143 RPN140 RPN141 RPN144
PS
Protein A Anti-mouse Anti-rabbit
RPNQ0264 RPNQ0265 RPNQ0298 RPNQ0299
YOx
Protein A Anti-mouse Anti-rabbit
RPNQ0274 RPNQ0275 RPNQ0300 RPNQ0301
CCD imagers
Bulk order quantities are available for larger scale research experiments and high-throughput screens.
For a range of fully optimized scintillation proximity radioimmunoassay kits, please visit our Web sites at www.gehealthcare.com/spa and www.gehealthcare.com/leadseeker. A full listing of SPA beads is shown on page 23.
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Application of SPA technology to cell-based assays GE Healthcare has developed Cytostar-T™ Scintillating Microplates specifically for the study of cell-based assays using SPA technology, which offer a number of benefits.
Fig 11. Section of a single well from a Cytostar-T Scintillating Microplate illustrating the principle of Cytostar-T.
Versatile • A range of applications from cell proliferation and metabolite transport to mRNA quantitation; • Uses a range of beta-emitting isotopes such as 14C and 33P
Homogeneous • Real time and non-invasive quantitation • Amenable to automation
Tissue culture compatible • Includes sterile and tissue-culture treated plate with lid • Suitable for culture of adherent cell lines • Transparent base allows visualization of cells • 96-well plate format • Same size and shape as traditional microplates.
Convenient • Scintillant incorporated into base plates • No liquid scintillant to add • No liquid scintillant disposal costs.
Compatible with existing instrumentation • Wallac MicroBeta™ scintillation counter • Packard TopCount™ scintillation counter • Hidex Chameleon™ plate reader 18
Principle of Cytostar-T Scintillating Microplates Cytostar-T Scintillating Microplates contain a scintillant incorporated into the base plate and are tissue culture treated and sterilized. The wells have transparent bases allowing adherent cells that are cultured on the plates to be visualized. The close proximity of the adherent cells to the base plate means that any isotope taken up by the cells from the surrounding media generates a light signal. Any isotope free in solution is too far removed from the scintillant in the base plate to stimulate the scintillant and no signal is generated. This means that Cytostar-T Scintillating Microplates can be utilized in non-invasive and real time analysis of cellular events. The plates can be detected on standard plate-based scintillation counters, so no specialist instrumentation is required.
Ordering information Product
Quantity
Product code
Cytostar-T Scintillating Microplates
5 x 96 wells
RPNQ0162
Cytostar-T Scintillating Microplates
100 x 96 wells
RPNQ0163
For further information on Cytostar-T Scintillating Microplates, please visit our Web site at www.gehealthcare.com/cytostar.
19
Application of SPA to drug metabolism and pharmacokinetics DMPK assays are a range of kits for use in drug metabolism and pharmacokinetic studies. Based on SPA technology, DMPK assays provide an economical and higher throughput alternative to conventional assay methodologies such as equilibrium dialysis, HPLC, and ultrafiltration. • High-throughput screening: homogeneous assay formats can be readily miniaturized for use in automated systems • Range of applications: choice of absorption, distribution, metabolism, and toxicology assays • Fully validated: developed using panels of compounds with binding profiles determined by equilibrium dialysis and ultrafiltration
Ordering information Product
Quantity
Product code
Alpha 1-Acid Glycoprotein (AGP) [3H] SPA Binding Assay
5 x 96 wells
TRK1090-40μCi
Cytochrome P450 (CYP2D6) [ C] SPA Activity Assay
5 x 96 wells
CFA772-12μCi
Cytochrome P450 (CYP3A4) [3H] SPA Competition Binding Assay
5 x 96 wells
TRK1091-15μCi
High-Density Lipoprotein (HDL) [3H] SPA Binding Assay
5 x 96 wells
TRK1092-25μCi
Human Serum Albumin (HSA) [3H] SPA Binding Assay
5 x 96 wells
TRK1093-45μCi
Leucine Uptake [14C] Cytostar-T Assay
5 x 96 wells
CFA773-50μCi
Low-Density Lipoprotein (LDL) [ H] SPA Binding Assay
5 x 96 wells
TRK1094-25μCi
Thymidine Uptake [14C] Cytostar-T Assay
5 x 96 wells
CFA775-50μCi
14
3
All DMPK Assays are made to order, please inquire.
Receptor Membrane Preparations GE Healthcare provides a range of recombinant GPCR membrane receptor preparations ready for use and ideal for applications such as high-throughput screening, profiling, cross-reactivity testing, and GTPγS assays. The receptors available include the following: • Adenosine
• Cholecystokinin
• Neurokinin
• Adrenergic
• Dopamine
• Neurotensin
• Angiotensin
• Endothelin
• Nociceptin
• Apelin
• Histamine
• Opioid
• Cannabinoid
• Galanin
• Serotonin
• CGRP
• Motilin
• Vasopressin
• Chemokine
• Muscarinic
Standard pack size is 200 U. Larger quantities are available on request. Please inquire for details. 20
Scintillation counter settings SPA Scintillation Beads can be counted on a range of scintillation counters. For counters fitted with spectrum analysis packages, the suitable window opening can be determined and the instrument set accordingly (Table 10). For further information regarding programming and calibration of counters, please contact your instrument manufacturer. Table 10. Settings for counting SPA in commonly used scintillation counters.
Counter type
Isotope and SPA bead type 3
125
H
PVT
YSi
PVT
33
I YSi
PVT
35
P YSi
14
S
PVT
YSi
C
PVT
YSi
Packard TopCount
1.5–35
0–50
1.5–100 0–100
2.9–256 2.9–256 2.9–256 2.9–256 2.9–256 2.9–256
Wallac MicroBeta
5–360
5–500
5–530
5–650
Hidex Chameleon*
20–120 20–120 20–900 20–900 20–600 20–600 N.D
Beckman LS7800
H# is set at 0 and the window is wide open from 10 to 999
Packard Tricarb 460
Window open from 0 to 999
5–650
5–750
5–600
5–700
5–650
5–750
N.D.
N.D.
N.D.
LKB 1209/1215 RackBeta Window open from 5 to 999 * These counter settings are not fully optimized. N.D. = Not determined
Multiwell plates suitable for counting SPA Scintillation Beads A range of multiwell plates is suitable for counting signal emitted by SPA Scintillation Beads. Table 11 provides descriptions, suppliers and codes of the most commonly used plates plus information on which counter is most suitable. Table 11. Multiwell plates suitable for use with SPA Scintillation Beads
Plate type and description
Supplier and code
Dynatech Microlite 1, white, solid base
Dynatech
No. of wells 96
Packard TopCount
Counter
Corning, non-binding Surface (NBS), white, solid base
Corning 3600
96
Packard TopCount
Corning, non-binding Surface (NBS), white, solid base
Corning 3604
96
Wallac MicroBeta
Corning, non-treated, white, solid base
Corning 3365
96
Packard TopCount
Corning, non-treated, white, clear base
Corning 3632
96
Wallac MicroBeta
Isoplate, white, clear base
Perkin Elmer 1450-514
96
Wallac MicroBeta
Optiplate, white, solid base
Perkin Elmer 6005207
96
Packard TopCount
Optiplate, white, solid base*
Perkin Elmer 6005256
384
Packard TopCount
Corning, non-treated, white, solid base
Corning 3705
384
Packard TopCount
Corning, non-treated, white, clear base
Corning 3706
384
Wallac MicroBeta
Greiner, white, solid base
Greiner 781075
384
Packard TopCount
*
May not have been used by GE Healthcare but the manufacturer claims the plates are suitable for SPA.
21
Multiwell plates suitable for measuring SPA Imaging Beads A range of multiwell plates is suitable for measuring signal emitted by SPA Imaging Beads. Table 12 provides descriptions, suppliers and codes of the most commonly used plates plus information on which imager is most suitable. Table 12. Multiwell plates suitable for use with SPA Imaging Beads.
Plate type and description
Supplier and code
No. of wells
Imager
Nunc, polystyrene, solid, white, untreated 1536-well plate
Nunc 253607
1536
LEADseeker
Nunc, polystyrene, solid, white, untreated 384-well plate
Nunc 264572
384
LEADseeker
Nunc, low cross talk, polypropylene, solid, white, untreated 384-well plate
Nunc 264675
384
LEADseeker
Nunc, 96-microwell plate, polystyrene, solid, white, untreated
Nunc 236108
96
LEADseeker
Greiner, 384-well microplate, polystyrene, LUMITRAC 200, solid, white, medium binding
Greiner 781075
384
LEADseeker
Greiner, 384-well microplate, polystyrene, Gen I small volume, solid, white, medium binding
Greiner 784075
384
LEADseeker
Greiner, 384-well microplate, polystyrene, Gen II small volume, solid, white, medium binding
Greiner 788075
384
LEADseeker
Greiner, HiBase 1536-well microplate, polystyrene, solid, white, LUMITRAC 200, medium binding
Greiner 782075
1536
LEADseeker
Greiner, LoBase 1536-well microplate, polystyrene, solid, white, LUMITRAC 200, medium binding
Greiner 783075
1536
LEADseeker
Greiner, 96-well microplate, polystyrene, F-bottom, solid, white, LUMITRAC 200, medium binding
Greiner 655075
96
LEADseeker
Corning, Non-binding surface (NBS), white, solid base
Corning 3600
96
LEADseeker
Corning, non-treated, white, solid base
Corning 3365
96
LEADseeker
Corning, non-treated, white, solid base
Corning 3705
384
LEADseeker
Corning, 1536 2-μl plates, non-treated, white, solid base
Corning 3852
1536
LEADseeker
Corning, 1536 10-μl non-treated, white, solid base
Corning 3937
1536
LEADseeker
Corning, TC-treated, sterile, white, solid base
Corning 3704
384
LEADseeker
Optiplate, white, solid base
Perkin Elmer 6005256
384
LEADseeker
Optiplate, white, solid base
Perkin Elmer 6005207
96
LEADseeker
Dynatech Microlite 1, white, solid base
Dynatech
96
LEADseeker
22
Ordering information Coating
Core bead type
Pack size
Streptavidin
PVT
50 mg 150 mg 500 mg 25 x 500mg 75 mg 250 mg 500 mg 50 mg 25 x 500 mg 500 mg 50 mg 25 x 500 mg 100 mg 500 mg 25 x 500 mg 250 mg 500 mg 50 mg 25 x 500 mg 500 mg 50 mg 25 x 500 mg 500 mg 500 mg 50 mg 500 mg 500 mg 50 mg 500 mg 500 mg 50 mg 1000 mg 500 mg 50 mg 500 mg 500 mg 500 mg 250 mg 125 mg 500 mg 50 mg 500 mg 50 mg 500 mg 50 mg 500 mg 500 mg 500 mg 50 mg 500mg 50mg 500mg 500mg 500 mg 500 mg
YSi PS YOx WGA
PVT YSi PS YOx
WGA PEI Type A WGA PEI Type B PEI (polyethyleneimine) Poly-L-lysine Membrane binding beads PDE Arginine binding beads Copper chelate Nickel chelate
PVT PS PVT PS PVT PS YSi PS YOx YSi YSi PVT YSi PS YOx
Glutathione
YSi
Protein A
PVT YSi YOx PS
Anti-mouse antibody
PVT YSi PS YOx
Product code SPA Scintillation SPA Imaging Beads Beads RPNQ0006 RPNQ0009 RPNQ0007 SPQ0032 RPNQ0015 RPNQ0012 RPNQ0261 RPNQ0263 RPNQ0285 RPNQ0271 RPNQ0273 RPNQ0283 RPNQ0252 RPNQ0001 SPQ0031 RPNQ00011 RPNQ0260 RPNQ0262 RPNQ0281 RPNQ0270 RPNQ0272 RPNQ0282 RPNQ0003 RPNQ0287 RPNQ0286 RPNQ0004 RPNQ0289 RPNQ0288 RPNQ0097 RPNQ0098 RPNQ0297 RPNQ0010 RPNQ0294 RPNQ0295 RPNQ0280 RPNQ0150 RPNQ0101 RPNQ0095 RPNQ0096 RPNQ0266 RPNQ0267 RPNQ0276 RPNQ0277 RPNQ0034 RPNQ0033 RPNQ0019 RPN143 RPNQ0274 RPNQ0275 RPNQ0264 RPNQ0265 RPNQ0017 RPN141 RPNQ0298 RPNQ0300 23
Coating
Core bead type
Pack size
Anti-rabbit antibody
PVT YSi PS YOx PVT YSi YOx PVT and YSi PS and YOx
500 mg 500 mg 500 mg 500 mg 500 mg 500 mg 500 mg
Anti-sheep antibody Charge-based binding SPA Scintillation Select-a-Bead Kit* SPA Imaging Select-a-Bead Kit†
Product code SPA Scintillation SPA Imaging Beads Beads RPNQ0016 RPN140 RPNQ0299 RPNQ0301 RPNQ0018 RPN142 RPNQ0320 RPNQ0250 RPNQ0291
* Contains 100 mg each of WGA PVT; WGA YSi; WGA PEI Type A, WGA PEI Type B, and Poly-L-lysine to allow quick and convenient receptor assay development using SPA Scintillation Beads. † Contains 50 mg each of WGA PS; WGA YOx; WGA PEI Type A, WGA PEI Type B, and Poly-L-lysine to allow quick and convenient receptor assay development using SPA Imaging Beads.
Related products These SPA screening kits are designed for use with SPA Scintillation Beads and do not contain enzyme unless specifically stated. Product
Quantity
Contents
Cholesterol Ester Transfer Protein (CETP) [3H] SPA (includes enzyme)
500 assay points
Cholesterol Ester Transfer Protein (CETP) [3H] SPA
500 assay points
[3H] PVT SPA Color Quench Kit
Sufficient for one uniformity check or one color quench correction curve.
• [3H]Cholesteryl ester HDL TRKQ7005-25UCI • Stop reagent (contains SPA Scintillation Beads) • Biotin LDL • Assay buffer • CETP (human) • Protocol • [3H]Cholesteryl ester HDL TRKQ7015-25UCI • Stop reagent (contains SPA Scintillation Beads) • Biotin LDL • Assay buffer • Protocol • [3H]PVT SPA Scintillation Beads TRKQ7080 • Tartrazine solution • Protocol
[125I] PVT SPA Color Quench Kit
Sufficient for one uniformity check or one color quench correction curve.
• [125I]PVT SPA Scintillation Beads • Tartrazine solution • Protocol
Farnesyl Transferase [3H] SPA Enzyme Assay
500 assay points
Helicase [3H] SPA Enzyme Assay
500 assay points
• Assay buffer TRKQ7010-100UCI • Bead reconstitution buffer • Biotin lamin B sequence peptide • Stop reagent • [3H]Farnesyl pyrophosphate (100 μCi) • Streptavidin SPA Scintillation Beads • Protocol • [3H]Helicase substrate solution (40 μCi) TRKQ7030-40UCI • Stop/capture solution • Streptavidin Scintillation SPA Beads in diluent buffer • Substrate dilution buffer • Assay buffer • Protocol
24
Product code
RPAQ4030-56UCi
Product
Quantity
Contents
Product code
HIV-1 Integrase [3H] SPA Enzyme Assay
500 assay points
TRKQ7020-60UCI
Nitric Oxide Synthase Screening Kit
500 assay points
p34cdc2 [33P] Protein Kinase Enzyme Assay
500 assay points
Phosphodiesterase [3H]cAMP SPA Enzyme Assay
500 assay points
Phosphodiesterase [3H]cGMP SPA Enzyme Assay
500 assay points
Phospholipase A 2 [3H] SPA
500 assay points
[3H] Quan-T-RT
200 assay points
Serine/Threonine Protein Kinase SPA [33P] Enzyme Assay
500 assay points
Thymidine Uptake Assay
500 assay points
Biotinylated Myelin Basic Protein (bMBP)
25 μM
• [3H]DNA oligonucleotide • Denaturing solution • Assay buffer • Streptavidin SPA Scintillation Beads (lyophilized) • Manganese chloride solution • Bead reconstitution solution • Stop solution • Protocol • [3H]Arginine (70 μCi) • Arginine binding YSi SPA Scintillation Beads • Protocol • Assay buffer (includes biotin-tagged peptide) • Streptavidin PVT SPA Scintillation Beads • Stop solution • Protocol • [3H]cAMP • SPA Scintillation Beads • Assay buffer • Protocol • [3H]cGMP • SPA Scintillation Beads • Assay buffer • Protocol • Phospholipase A2 [3H] SPA substrate • Phospholipid mixed micelles • Assay buffer • Stop reagent/bead resuspension buffer • Streptavidin SPA Scintillation Beads • Protocol • Annealed primer/ template, bound to • Streptavidin SPA Scintillation Beads • Assay buffer • Stop solution • [3H]TTP (100 μCi) • Protocol • Assay buffer (including biotinylated substrate • Streptavidin PVT SPA Scintillation Beads • Stop solution • Protocol • SPA Scintillation Beads • Lysis buffer • Enhancer • Protocol • Supplied frozen, in buffer
100 μM
TRKQ7160-70UCI
RPNQ0170
TRKQ7090
TRKQ7100
TRKQ7040
TRK1022
RPNQ0200
RPNQ0130
RPNQ0201-2.5ml RPNQ0201-10ml RPNQ0202-1ml* RPNQ0202-10ml RPNQ0202-50ml
*bMBP: 1 ml is typically sufficient for ~ 200 assay points.
25
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www.gehealthcare.com GE Healthcare Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK
General Electric Company reserves the right, subject to any regulatory approval if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your GE Representative for the most current information. © 2005 General Electric Company - All rights reserved. GE and GE Monogram are trademarks of General Electric Company. LEADseeker, Cytostar-T, Amersham, and Amersham Biosciences are trademarks of GE Healthcare Limited. Cytostar-T is covered by US patent numbers 5665562 and5989854, European patent number 650396 B1, Japanese patent number 3152933, Finnish patent number 110488, and Canadian patent application number 2139409.Scintillation proximity assay technology is covered by European patent number 0154734 and by Japanese patent application number 84/52452. SPA technology using Imaging Beads is protected by Amersham Biosciences under US Patent Number 6,524,786, and foreign equivalents. MicroBeta and TopCount are trademarks of Perkin Elmer Life Sciences. Chameleon is a trademark of Hidex Oy. Amersham Biosciences UK Ltd, a General Electric company going to market as GE Healthcare.
imagination at work 28-4074-97 AA
SPA product guide
Amersham SPA product guide Contents Scintillation proximity assay—versatile technology for rapid and sensitive assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 The principle of SPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Advantages of SPA bead-based assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Choosing between SPA Scintillation Beads and SPA Imaging Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 SPA bead types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 SPA bead coatings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 Suitable radioisotopes for SPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 Application of SPA technology to enzyme assays
. . . . . . . . . . . . . . . . . . . . . . . .7
Assay format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 SPA bead types and coatings used for enzyme assays . . . . . . . . . . . . . . . . . . . .8 Application of SPA technology to kinase assays
. . . . . . . . . . . . . . . . . . . . . . . . . .9
SPA bead types and coatings used for kinase assays . . . . . . . . . . . . . . . . . . . . . .9 Application of SPA technology to receptor-binding assays . . . . . . . . . . .11 SPA bead types and coatings for receptor-binding assays . . . . . . . . . . . . . .12 Typical applications for receptor-binding SPAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 Application of SPA technology to molecular interaction research . .14 SPA bead types and coatings for studying molecular interactions
. . . .14
Application of SPA technology to radioimmunoassays . . . . . . . . . . . . . . . .16 Application of SPA technology to cell-based assays . . . . . . . . . . . . . . . . . . . .17 Principle of Cytostar-T Scintillating Microplates
. . . . . . . . . . . . . . . . . . . . . . . . . . . .18
Application of SPA technology to drug metabolism and pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 Receptor Membrane Preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 Scintillation counter settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 Multiwell plates suitable for counting SPA Scintillation Beads . . . . . . .20 Multiwell plates suitable for measuring SPA Imaging Beads
. . . . . . . . .21
Ordering information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22 Related products
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
1
Scintillation proximity assay—versatile technology for rapid and sensitive assays Scintillation proximity assay (SPA) is a homogeneous and versatile technology for the rapid and sensitive assay of a wide range of biological processes. The assay format requires no separation steps, is amenable to automation, and is supported by more than 10 years of application data generated independently and by GE Healthcare scientists. SPAs using either SPA Scintillation Beads or SPA Imaging Beads can be read on scintillation counters and CCD imagers respectively.
Versatile A range of bead types and coatings • For use with enzyme and receptor targets, radioimmunoassays, and molecular interactions • Various pack sizes available from stock for assay development and research • Bulk packs available for screening applications • Custom bead coatings on request SPA beads are suitable for use with a range of radioisotopes • 3H, 125I, 33P, and 14C can be used as radiolabels • No requirement for protein modification Compatible with any scintillation counter or CCD imager • SPA Scintillation Beads can be used in tubes and 96- or 384-well microplates • SPA Imaging Beads can be used in any format microplate
No separation step Reduction in handling steps • Increased assay precision and assay reproducibility • Improved safety • No need for filters and scintillation cocktails Reduction in radioactive liquid waste • Decreased disposal costs Amenable to automation • Increased throughput
Proven track record • Leading radiometric screening technology • Supported by more than 10 years of application data generated by GE Healthcare and leading pharmaceutical companies • Over 600 literature references are available on our Web sites at www.gehealthcare.com/spa and www.gehealthcare.com/leadseeker.
2
The principle of SPA When certain radioactive atoms decay, they release ß-particles. The distance these particles travel through an aqueous solution is dependent on the energy of the particle. If a radioactive molecule is held in close enough proximity to a SPA Scintillation Bead or a SPA Imaging Bead, the decay particles stimulate the scintillant within the bead to emit light, which is then detected in a PMT-based scintillation counter or on a CCD-based imager respectively (Table 1). However, if a radioactive molecule is free in a solution containing SPA beads, the decay particles will not have sufficient energy to reach the bead and no light will be emitted. This discrimination of binding by proximity means that no separation of bound and free ligand is required.
Free radiolabel
Bound radiolabel
Fig 1. SPA principle.
Advantages of SPA bead-based assays SPA bead-based assays have many advantages over plate-based methods • The higher surface area of SPA beads allows the concentration of key assay components (e.g. receptor membrane) to be increased or decreased, thus giving greater flexibility in assay design. • The higher binding capacity of SPA beads gives higher signal allowing a potential reduction in the amount of radioactive label required. • The amount of SPA bead can be altered to optimize the sensitivity of an assay. • SPA beads are format independent making it easier to transfer assays to higher density plates. • A choice of four different core bead matrices allows optimization of signal and background over a wide range of applications (Table 2). • SPA beads are not dependent on one plate type or supplier (Table 11).
3
Choosing between SPA Scintillation Beads and SPA Imaging Beads The choice of SPA bead depends on several parameters and these are summarized in Table 1. Table 1. SPA bead selection guide
SPA Scintillation SPA Imaging Beads Beads
Feature
Benefit
PMT readers, standard beta counters
CCD Imagers
SPA Scintillation Beads emit in the blue region and SPA Imaging Beads emit light in the red region of spectra (Fig 2).
Match bead to instrument type (e.g. CCD imagers are more sensitive in the red region).
Throughput Medium to high
High to ultra high
Choice of throughput
Match instrument and bead choice with throughput needs
Format
96- or 384well plates
96-, 384-, 1536-well Choice of format plates and above
Increase throughput and decrease assay component costs
Emission
Blue region (400 nm)
Red region (615 nm)
Reduce color quench caused by orange/red colored compounds through use of SPA Imaging Beads
Instrument
Choice of emission properties
Different colored dyes can affect the signal produced in a SPA using either SPA Scintillation Beads or SPA Imaging Beads (Fig 3). The dyes used to generate the data in Figure 3 were at concentrations of up to 3 mg/ml, which is in excess of that liable to be found in typical compound libraries, while all wells have identical amounts of radiolabel and SPA bead. The data show no significant attenuation of signal from the SPA Imaging Beads at any concentration of tartrazine, whereas the SPA Scintillation Beads report a lower level of signal as dye concentration increases. The presence of Chicago Sky Blue (CSB) causes quenching of the SPA Imaging Bead signal from a concentration of 0.03 mg/ml; the YSi and PVT beads only begin to show quenching at 0.1 mg/ml of CSB. Given that there is a higher proportion of yellow/orange/red compounds than blue compounds in most compound libraries, the issue of color quenching is reduced by conversion to SPA Imaging Beads.
4
Relative intensity
Fig 2. Wavelength emissions from SPA Scintillation Beads (PVT and YSi) and SPA Imaging Beads (PS and YOx ). The overlaid spectrum indicates the compound colors that have the potential to quench bead signal.
Wavelength (nM)
Tartrazine
Fig 3. Plate map showing effect of color quenching on an imaged assay, on SPA Scintillation Beads (PVT and YSi), and SPA Imaging Beads (PS and YOx).
Chicago Sky Blue
SPA bead types SPA Scintillation Beads and SPA Imaging Beads are available in different core materials and their benefits are described in Table 2. Table 2. SPA bead type selection guide.
SPA Scintillation Beads SPA Imaging Beads
Benefit
Polyvinyltoluene (PVT)
Polystyrene (PS)
Easy to dispense and suited to automation and high-throughput screening. Used for the majority of SPA scintillation and SPA imaging applications
Yttrium silicate (YSi)
Yttrium oxide (YOx)
Possess greater scintillation counting efficiency but less suited to automation because their higher density makes them settle more rapidly than PVT or PS beads
5
SPA bead coatings SPA beads are coated with coupling molecules such as wheat germ agglutinin (WGA), streptavidin, antibodies, glutathione, and copper or nickel chelate. These coupling molecules allow a variety of assay reagents such as cell membranes, biotinylated peptides, and fusion proteins to be captured onto the bead. The choice of bead coating will be determined by the application under investigation. Table 3 gives guidance on which bead coating is most suited to a range of typical applications. Table 3. SPA bead coatings and their recommended applications.
Bead coating
Bead types available
Application
Arginine Binding
YSi
Capture of arginine
Charge-Based Binding
YOx
Capture of GST fusion substrates
Copper chelate (His-Tag) Nickel Chelate (His-Tag) Glutathione Streptavidin
PVT,YSi PS, YOx YSi PVT, YSi, PS, YOx,
Enzyme assays, molecular interactions
Wheat Germ Agglutinin (WGA) Membrane Binding Polyethyleneimine (PEI) Polylysine Select-a-Bead Kit*
PVT, YSi, PS, YOx YOx PS, PVT PS, YSi PVT, YSi, PS, YOx
Receptor-binding
Anti-Mouse Anti-Rabbit Anti-Sheep Protein A
PVT, YSi, PS, YOx PVT, YSi, PS, YOx PVT, YSi PVT, YSi, PS, YOx
RIA, enzyme assays, molecular interactions
PDE
YSi
PDE activity
Suitable radioisotopes for SPA The most important characteristic when selecting a radioisotope for SPA is the pathlength of the decay particle (Table 4). In general, the shorter the pathlength of the decay particle, the more suited it is to SPA. • Tritium and iodine-125 are ideally suited to SPA • Carbon-14, sulfur-35, and phosphorus-33 have been used successfully with SPA Table 4. Radioisotopes suitable for SPA and their decay particle average pathlengths.
Isotope
Average pathlength of decay particle in aqueous solution
Tritium
1.5 μm
Iodine-125
2e-: 1.0 μm, 17 μm
Carbon-14
50 μm
Sulfur-35
65 μm
Phosphorus-33
125 μm
6
Application of SPA technology to enzyme assays Enzyme SPA applications can be used to identify potential new enzyme inhibitors, measure enzyme activity, and perform kinetic analysis. Activity of the following enzymes can be determined by enzyme SPA . • Hydrolases such as proteases and nucleases • DNA modifying enzymes such as helicase and integrase • Transferases • Phosphodiesterases • Lipid modifying enzymes Examples of enzyme SPA applications can be found as Proximity News articles on our SPA Web site at www.gehealthcare.com/spa and as Leads articles on our LEADseeker™ Web site at www.gehealthcare.com/leadseeker. These Web sites also include bibliographies containing further publications citing enzyme SPA applications.
Assay format The format of an enzyme SPA depends on the action of the enzyme being studied. 1. Signal increase assays are used to measure the activity of kinases, polymerases, and transferases because these enzymes add radiolabeled molecules to the substrate immobilized on the bead (Fig 4a). 2. Signal decrease assays are used to measure the activity of enzymes such as proteases and nucleases because they cleave radiolabeled substrate from the bead (Fig 4b).
a.
b.
Fig 4. Enzyme SPA concept. (a) Signal increase format (b) Signal decrease format.
7
SPA bead types and coatings used for enzyme assays 1. The majority of enzyme SPAs use biotinylated substrates that may be either immobilized on, or subsequently captured by, streptavidin-coated SPA Scintillation Beads or SPA Imaging Beads. The biotin-streptavidin system provides a reliable and reproducible high-affinity capture mechanism. Further details on biotinylation techniques can be found in Proximity News 39, a copy of which is available on our SPA Web site at www.gehealthcare.com/spa. 2. Arginine Binding YSi SPA Beads are used in the Nitric Oxide Synthase (NOS) Screening Kit (TRKQ7160). Figure 5 shows timecourse data whereby [3H]arginine is converted to citrulline by the action of NOS. The product, [3H]citrulline, does not bind to the beads and the action of NOS is therefore observed as a reduction in SPA counts.
Fig 5. Timecourse data for the Nitric Oxide Synthase (NOS) Screening Kit.
3. GST-tagged fusion protein enzyme substrates can be captured onto SPA beads coated with glutathione. 4. His-tagged fusion proteins can be captured onto copper chelate coated SPA Scintillation Beads, nickel chelate coated SPA imaging Beads or charge based binding SPA Imaging Beads. Table 5. SPA bead types and coatings commonly used in enzyme SPA applications.
Instrument
Core bead type
Coating
Product code
PMT readers
PVT
Streptavidin Copper chelate
RPNQ0006, RPNQ0007, RPNQ0009 RPNQ0095
YSi
Streptavidin Copper chelate Glutathione PDE Arginine binding SPA beads
RPNQ0012, RPNQ0015 RPNQ0096 RPNQ0033, RPNQ0034 RPNQ0150 RPNQ0101
PS
Streptavidin Nickel chelate
RPNQ0261, RPNQ0263 RPNQ0266, RPNQ0267
YOx
Streptavidin Nickel chelate Charge-based binding
RPNQ0271, RPNQ0273 RPNQ0276, RPNQ0277 RPNQ0320
CCD imagers
Bulk order quantities are available for larger scale research experiments and high-throughput screens.
For a range of fully optimized enzyme SPA kits, please visit our Web site at www.gehealthcare.com/spa. A full listing of SPA beads is shown on page 23. 8
Application of SPA to kinase assays Kinases play pivotal roles in many signal transduction cascades and consequently kinase activities remain a key focus of academic and pharmaceutical research. GE Healthcare has developed kinase SPA applications for tyrosine and serine/threonine kinases using both peptide and protein substrates. • Cyclin dependent kinases
• MAP kinases
• Protein kinase A
• Protein kinase C isoforms
The basic principle of kinase SPA involves the transfer of [33P]phosphate from [33P]ATP to the tyrosine, serine, or threonine in the peptide or protein substrate. The phosphorylated peptide is then captured by a streptavidin-coated SPA bead (Fig 6).
Kinase enzyme
Biotinylated peptide or MBP [33P]-ATP substrate
Streptavidin SPA Imaging Beads
ADP
Fig 6. Kinase SPA concept.
SPA bead types and coatings used for kinase assays Typically, kinase SPAs are configured by using streptavidin-coated SPA beads and biotinylating the peptide or protein substrate. Alternatively, fusion protein binding SPA beads can also be used. A list of kinase applications can be found in the bibliography section on our Web sites at www.gehealthcare.com/spa and www.gehealthcare.com/leadseeker.
Fig 7. Inhibition of p56lck kinase activity by PP2 and nocodazole in 1536-well format using Polystyrene SPA Imaging Beads. Each data point is the mean ± SEM of six replicates.
9
Table 6. SPA bead types and coatings commonly used in kinase SPA applications.
Instrument
Core bead type
Coating
Product code
PMT readers
PVT
Streptavidin Copper chelate
RPNQ0006, RPNQ0007, RPNQ0009 RPNQ0095
YSi
Streptavidin Copper chelate
RPNQ0012, RPNQ0015 RPNQ0096
PS
Streptavidin Nickel chelate
RPNQ0261, RPNQ0263 RPNQ0266, RPNQ0267
YOx
Streptavidin Nickel chelate Charge-based binding
RPNQ0271, RPNQ0273 RPNQ0276, RPNQ0277 RPNQ0320
CCD imagers
Bulk order quantities are available for larger scale research experiments and high-throughput screens.
For a range of fully optimized enzyme SPA kits, please visit our Web site at www.gehealthcare.com/spa. A full listing of SPA beads is shown on page 23.
10
Application of SPA technology to receptor-binding assays Receptor-binding SPAs can be configured to determine receptor kinetics, saturation binding, or to detect inhibitors of radioligand binding. SPA has been successfully applied to receptor-binding assays by immobilizing receptors directly to SPA beads via a number of coupling methods. SPA receptor-binding assays have been developed for a wide range of receptor types, including: • Serotonin • Acetylcholine • Chemokine • Cytokine • Steroid • Inositol triphosphate SPA technology also enables the binding of GTP to G-protein coupled receptors (GPCRs) following agonist binding to the GPCR to be measured. SPA is compatible with receptors prepared from a number of different sources: • Cell membrane preparations from tissue • Cell membrane preparations from cultured cells • Soluble purified receptors • Solubilized receptors from tissues and cultured cells The principle of receptor-binding SPAs involves radiolabeled ligand binding to a receptor immobilized on the surface of a SPA bead. The bound ligand is held in close enough proximity to the bead to stimulate scintillant within the bead to emit light. Unbound radioligand is too distant from the bead to transfer energy and therefore goes undetected (Fig 8).
Bead emitting light Fig 8. Receptor-binding SPA principle.
11
SPA bead types and coatings used for receptor-binding assays 1. Receptor-binding SPAs are usually formatted using wheat germ agglutinin (WGA)-coated SPA beads. WGA binds N-acetyl-ß-D-glucosaminyl residues, N-acetyl-ß-D-glucosamine oligomers, and glycoproteins present in cell membranes to capture cell membranes expressing the receptor of interest. 2. The use of polyethyleneimine to either treat WGA or as a separate bead coating can further reduce non-specific binding, 3. Poly-L-lysine is used for binding negatively charged cellular membranes. 4. Soluble and solubilized receptors can be captured onto antibody binding SPA beads via an antibody specific to the receptor. 5. The receptor protein can be biotinylated and captured onto streptavidin-coated beads.
Typical applications for receptor-binding SPAs Proximity News, 27, describes the steps required to develop a receptor-binding SPA and can be found on our SPA Web sites at www.gehealthcare.com/spa and www.gehealthcare.com/leadseeker. Further examples of receptor-binding SPA applications can be found as Proximity News articles on the SPA Web site, which also includes a bibliography containing over 170 publications citing receptor-binding SPA applications. Application notes detailing receptor-binding applications using SPA Imaging Beads can be found at www.gehealthcare.com/spa. Data from a typical receptor-binding assay is shown in Figure 9 whereby competition analysis was performed on membranes from HEK cells expressing the human melanocortin 4 receptor. The radiolabeled ligand, [125I]NDP-α-MSH - [Nle4-D-Phe] melanocyte stimulating hormone and the competitor (unlabelled NDP-α-MSH) were added together with WGA-coated Polystyrene SPA Imaging Beads at T = 0 into the wells of 384-well plates. The assays were incubated overnight at room temperature without shaking and the plates were then read for 5 min on the LEADseeker Multimodality Imaging System.
Fig 9. Melanocortin 4 receptor imaging SPA competition analysis. Values shown as means +/- SD (n = 3).
12
Table 7. SPA bead types and coatings that may be used in receptor-binding assays.
Instrument
Core bead type Coating
Product code
PMT readers
PVT
RPNQ0001, RPNQ0252 RPNQ0003 RPNQ0004 RPNQ0097 RPNQ0019, RPNQ0031, RPNQ0069 RPNQ0011 RPNQ0010 RPN143, RPNQ0068 RPNQ0250 RPNQ0260, RPNQ0262 RPNQ0286, RPNQ0287 RPNQ0288, RPNQ0289 RPNQ0098 RPNQ0264, RPNQ0265 RPNQ0294, RPNQ0295 RPNQ0274, RPNQ0275 RPNQ0291
YSi
CCD imagers
PVT and YSi PS
YOx PS and YOx
WGA WGA PEI Type A WGA PEI Type B Polyethyleneimine (PEI) Protein A WGA Poly-L-lysine Protein A SPA Select-a-Bead Kit* WGA WGA PEI Type A WGA PEI Type B PEI Protein A Poly-L-lysine Protein A SPA Imaging Select-a–Bead Kit†
*Contains 100 mg each of WGA PVT; WGA YSi; WGA PEI Type A, WGA PEI Type B, and poly-L-lysine to allow quick and convenient receptor assay development using SPA Scintillation Beads. †Contains 50 mg each of WGA PS; WGA YOx; WGA PEI Type A, WGA PEI Type B, and poly-L-lysine to allow quick and convenient receptor assay development using SPA Imaging Beads.
Bulk order quantities are available for larger scale research experiments and high-throughput screens. A range of fully optimized SPA kits for GPCR ligand binding are also available (see Ordering information) or visit our Web sites at www.gehealthcare.com/spa and www.gehealthcare.com/leadseeker. A full listing of SPA beads is shown on page 23.
13
Ordering information Product
Quantity
Code number
Adrenergic a2a [3H]SPA GPCR Assay
Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays Sufficient for 100 assays Sufficient for 400 assays
MRP0150-250UCI MRP0151-250UCI MRP0010-250UCI MRP0011-250UCI MRP0020-10UCI MRP0021-10UCI MRP0030-10UCI MRP0031-10UCI MRP0040-10UCI MRP0041-10UCI MRP0050-10UCI MRP0051-10UCI MRP0060-250UCI MRP0061-250UCI MRP0170-250UCI MRP0171-250UCI MRP0070-10UCI MRP0071-25UCI MRP0080-10UCI MRP0081-10UCI MRP0090-250UCI MRP0091-250UCI MRP0110-10UCI MRP0111-20UCI MRP0100-10UCI MRP0101-10UCI MRP0120-10UCI MRP0121-10UCI MRP0130-250UCI MRP0131-250UCI MRP0140-10UCI MRP0141-10UCI
Adrenergic a2c [3H]SPA GPCR Assay CCR6 [125I]SPA GPCR Assay CGRP [125I]SPA GPCE Assay CXCR2 [125I]SPA GPCR Assay CXCR3 [125I]SPA GPCR Assay Histamine H1 [3H]SPA GPCR Assay Histamine H3 [3H]SPA GPCR Assay Melanocortin MC4 [125I]SPA GPCR Assay Melanocortin MC5 [125I]SPA GPCR Assay Muscarinic M1 [3H]SPA GPCR Assay Neurokinin 2 [125I]SPA GPCR Assay Neuromedin NMU-1R [125I]SPA GPCR Assay Nociceptin ORL-1 [125I]SPA GPCR Assay Serotonin 5HT2c [3H]SPA GPCR Assay Somatostatin sst4 [125I]SPA GPCR Assay
14
Application of SPA technology to molecular interaction research SPA technology has been successfully applied to the study of a variety of molecular interactions. • Protein:protein interactions
• Protein:peptide interactions
• Protein:DNA interactions
• Cell adhesion molecule interactions
Many low-affinity binding events, such as occur between cell adhesion molecules, can be disrupted by the separation and wash steps required in heterogeneous techniques such as filtration. However, with SPA, these low-affinity binding events can be studied because there is no separation step.
SPA bead types and coatings for studying molecular interactions Various SPA bead types and coatings have been used for molecular interaction applications (Table 8): 1. Biotinylated peptides derived from SH2 binding proteins have been captured onto streptavidin-coated PVT SPA Scintillation Beads enabling the binding of a radiolabeled SH2 protein to the peptide to be observed. 2. Antibody capture assay formats utilizing antibody-binding SPA beads have been used to follow the binding of radiolabeled proteins, peptides, or in the case of transcription factors, DNA. 3. Glutathione and copper and nickel chelate coated fusion protein SPA Beads offer a more direct coupling method that does not require either additional reagents such as antibodies, or protein modification such as biotinylation. 4. Charge-based binding YOx SPA Imaging beads enable the capture and assay of glutathione S-transferase (GST) fusion proteins. The type of assay format feasible with the fusion protein binding SPA beads is shown in Figure 10.
Fig 10. Typical assay formats for molecular interaction SPA using glutathione or copper chelate coated SPA beads. 15
Table 8. SPA bead types and coatings commonly used in molecular interaction SPA applications.
Instrument
Core bead type
Coating
Product code
PMT readers
PVT
Protein A Anti-rabbit antibody Anti-mouse antibody Anti-sheep antibody Streptavidin Copper chelate
RPNQ0019 RPNQ0016 RPNQ0017 RPNQ0018 RPNQ0006, RPNQ0007, RPNQ0009 RPNQ0095
YSi
Protein A antibody Anti-Rabbit antibody Anti-mouse antibody Anti-sheep antibody Streptavidin Copper chelate Glutathione
RPN143 RPN140 RPN141 RPN142 RPNQ0012, RPNQ0015 RPNQ0096 RPNQ0033, RPNQ0034
PS
Streptavidin Nickel chelate
RPNQ0261, RPNQ0263 RPNQ0266, RPNQ0267
YOx
Streptavidin Nickel chelate Protein A Charge based binding
RPNQ0271, RPNQ0273 RPNQ0276, RPNQ0277 RPNQ0274, RPNQ0275 RPNQ0320
CCD imagers
A full listing of SPA beads is shown on page 23.
16
Application of SPA technology to radioimmunoassays Scintillation proximity radioimmunoassay is based on the reaction of antibody-bound ligand with SPA beads coated with either a secondary antibody or protein A. The range of SPA beads designed for use in RIA allows the conversion of heterogeneous RIA systems into the homogeneous SPA format. Table 9. SPA bead types and coatings that may be used in SPA radioimmunoassays.
Instrument
Core bead type
Coating
Product code
PMT readers
PVT
Protein A Anti-rabbit antibody Anti-mouse antibody Anti-sheep antibody
RPNQ0019 RPNQ0016 RPNQ0017 RPNQ0018
YSi
Protein A antibody Anti-rabbit antibody Anti-mouse antibody Anti-sheep antibody
RPN143 RPN140 RPN141 RPN144
PS
Protein A Anti-mouse Anti-rabbit
RPNQ0264 RPNQ0265 RPNQ0298 RPNQ0299
YOx
Protein A Anti-mouse Anti-rabbit
RPNQ0274 RPNQ0275 RPNQ0300 RPNQ0301
CCD imagers
Bulk order quantities are available for larger scale research experiments and high-throughput screens.
For a range of fully optimized scintillation proximity radioimmunoassay kits, please visit our Web sites at www.gehealthcare.com/spa and www.gehealthcare.com/leadseeker. A full listing of SPA beads is shown on page 23.
17
Application of SPA technology to cell-based assays GE Healthcare has developed Cytostar-T™ Scintillating Microplates specifically for the study of cell-based assays using SPA technology, which offer a number of benefits.
Fig 11. Section of a single well from a Cytostar-T Scintillating Microplate illustrating the principle of Cytostar-T.
Versatile • A range of applications from cell proliferation and metabolite transport to mRNA quantitation; • Uses a range of beta-emitting isotopes such as 14C and 33P
Homogeneous • Real time and non-invasive quantitation • Amenable to automation
Tissue culture compatible • Includes sterile and tissue-culture treated plate with lid • Suitable for culture of adherent cell lines • Transparent base allows visualization of cells • 96-well plate format • Same size and shape as traditional microplates.
Convenient • Scintillant incorporated into base plates • No liquid scintillant to add • No liquid scintillant disposal costs.
Compatible with existing instrumentation • Wallac MicroBeta™ scintillation counter • Packard TopCount™ scintillation counter • Hidex Chameleon™ plate reader 18
Principle of Cytostar-T Scintillating Microplates Cytostar-T Scintillating Microplates contain a scintillant incorporated into the base plate and are tissue culture treated and sterilized. The wells have transparent bases allowing adherent cells that are cultured on the plates to be visualized. The close proximity of the adherent cells to the base plate means that any isotope taken up by the cells from the surrounding media generates a light signal. Any isotope free in solution is too far removed from the scintillant in the base plate to stimulate the scintillant and no signal is generated. This means that Cytostar-T Scintillating Microplates can be utilized in non-invasive and real time analysis of cellular events. The plates can be detected on standard plate-based scintillation counters, so no specialist instrumentation is required.
Ordering information Product
Quantity
Product code
Cytostar-T Scintillating Microplates
5 x 96 wells
RPNQ0162
Cytostar-T Scintillating Microplates
100 x 96 wells
RPNQ0163
For further information on Cytostar-T Scintillating Microplates, please visit our Web site at www.gehealthcare.com/cytostar.
19
Application of SPA to drug metabolism and pharmacokinetics DMPK assays are a range of kits for use in drug metabolism and pharmacokinetic studies. Based on SPA technology, DMPK assays provide an economical and higher throughput alternative to conventional assay methodologies such as equilibrium dialysis, HPLC, and ultrafiltration. • High-throughput screening: homogeneous assay formats can be readily miniaturized for use in automated systems • Range of applications: choice of absorption, distribution, metabolism, and toxicology assays • Fully validated: developed using panels of compounds with binding profiles determined by equilibrium dialysis and ultrafiltration
Ordering information Product
Quantity
Product code
Alpha 1-Acid Glycoprotein (AGP) [3H] SPA Binding Assay
5 x 96 wells
TRK1090-40μCi
Cytochrome P450 (CYP2D6) [ C] SPA Activity Assay
5 x 96 wells
CFA772-12μCi
Cytochrome P450 (CYP3A4) [3H] SPA Competition Binding Assay
5 x 96 wells
TRK1091-15μCi
High-Density Lipoprotein (HDL) [3H] SPA Binding Assay
5 x 96 wells
TRK1092-25μCi
Human Serum Albumin (HSA) [3H] SPA Binding Assay
5 x 96 wells
TRK1093-45μCi
Leucine Uptake [14C] Cytostar-T Assay
5 x 96 wells
CFA773-50μCi
Low-Density Lipoprotein (LDL) [ H] SPA Binding Assay
5 x 96 wells
TRK1094-25μCi
Thymidine Uptake [14C] Cytostar-T Assay
5 x 96 wells
CFA775-50μCi
14
3
All DMPK Assays are made to order, please inquire.
Receptor Membrane Preparations GE Healthcare provides a range of recombinant GPCR membrane receptor preparations ready for use and ideal for applications such as high-throughput screening, profiling, cross-reactivity testing, and GTPγS assays. The receptors available include the following: • Adenosine
• Cholecystokinin
• Neurokinin
• Adrenergic
• Dopamine
• Neurotensin
• Angiotensin
• Endothelin
• Nociceptin
• Apelin
• Histamine
• Opioid
• Cannabinoid
• Galanin
• Serotonin
• CGRP
• Motilin
• Vasopressin
• Chemokine
• Muscarinic
Standard pack size is 200 U. Larger quantities are available on request. Please inquire for details. 20
Scintillation counter settings SPA Scintillation Beads can be counted on a range of scintillation counters. For counters fitted with spectrum analysis packages, the suitable window opening can be determined and the instrument set accordingly (Table 10). For further information regarding programming and calibration of counters, please contact your instrument manufacturer. Table 10. Settings for counting SPA in commonly used scintillation counters.
Counter type
Isotope and SPA bead type 3
125
H
PVT
YSi
PVT
33
I YSi
PVT
35
P YSi
14
S
PVT
YSi
C
PVT
YSi
Packard TopCount
1.5–35
0–50
1.5–100 0–100
2.9–256 2.9–256 2.9–256 2.9–256 2.9–256 2.9–256
Wallac MicroBeta
5–360
5–500
5–530
5–650
Hidex Chameleon*
20–120 20–120 20–900 20–900 20–600 20–600 N.D
Beckman LS7800
H# is set at 0 and the window is wide open from 10 to 999
Packard Tricarb 460
Window open from 0 to 999
5–650
5–750
5–600
5–700
5–650
5–750
N.D.
N.D.
N.D.
LKB 1209/1215 RackBeta Window open from 5 to 999 * These counter settings are not fully optimized. N.D. = Not determined
Multiwell plates suitable for counting SPA Scintillation Beads A range of multiwell plates is suitable for counting signal emitted by SPA Scintillation Beads. Table 11 provides descriptions, suppliers and codes of the most commonly used plates plus information on which counter is most suitable. Table 11. Multiwell plates suitable for use with SPA Scintillation Beads
Plate type and description
Supplier and code
Dynatech Microlite 1, white, solid base
Dynatech
No. of wells 96
Packard TopCount
Counter
Corning, non-binding Surface (NBS), white, solid base
Corning 3600
96
Packard TopCount
Corning, non-binding Surface (NBS), white, solid base
Corning 3604
96
Wallac MicroBeta
Corning, non-treated, white, solid base
Corning 3365
96
Packard TopCount
Corning, non-treated, white, clear base
Corning 3632
96
Wallac MicroBeta
Isoplate, white, clear base
Perkin Elmer 1450-514
96
Wallac MicroBeta
Optiplate, white, solid base
Perkin Elmer 6005207
96
Packard TopCount
Optiplate, white, solid base*
Perkin Elmer 6005256
384
Packard TopCount
Corning, non-treated, white, solid base
Corning 3705
384
Packard TopCount
Corning, non-treated, white, clear base
Corning 3706
384
Wallac MicroBeta
Greiner, white, solid base
Greiner 781075
384
Packard TopCount
*
May not have been used by GE Healthcare but the manufacturer claims the plates are suitable for SPA.
21
Multiwell plates suitable for measuring SPA Imaging Beads A range of multiwell plates is suitable for measuring signal emitted by SPA Imaging Beads. Table 12 provides descriptions, suppliers and codes of the most commonly used plates plus information on which imager is most suitable. Table 12. Multiwell plates suitable for use with SPA Imaging Beads.
Plate type and description
Supplier and code
No. of wells
Imager
Nunc, polystyrene, solid, white, untreated 1536-well plate
Nunc 253607
1536
LEADseeker
Nunc, polystyrene, solid, white, untreated 384-well plate
Nunc 264572
384
LEADseeker
Nunc, low cross talk, polypropylene, solid, white, untreated 384-well plate
Nunc 264675
384
LEADseeker
Nunc, 96-microwell plate, polystyrene, solid, white, untreated
Nunc 236108
96
LEADseeker
Greiner, 384-well microplate, polystyrene, LUMITRAC 200, solid, white, medium binding
Greiner 781075
384
LEADseeker
Greiner, 384-well microplate, polystyrene, Gen I small volume, solid, white, medium binding
Greiner 784075
384
LEADseeker
Greiner, 384-well microplate, polystyrene, Gen II small volume, solid, white, medium binding
Greiner 788075
384
LEADseeker
Greiner, HiBase 1536-well microplate, polystyrene, solid, white, LUMITRAC 200, medium binding
Greiner 782075
1536
LEADseeker
Greiner, LoBase 1536-well microplate, polystyrene, solid, white, LUMITRAC 200, medium binding
Greiner 783075
1536
LEADseeker
Greiner, 96-well microplate, polystyrene, F-bottom, solid, white, LUMITRAC 200, medium binding
Greiner 655075
96
LEADseeker
Corning, Non-binding surface (NBS), white, solid base
Corning 3600
96
LEADseeker
Corning, non-treated, white, solid base
Corning 3365
96
LEADseeker
Corning, non-treated, white, solid base
Corning 3705
384
LEADseeker
Corning, 1536 2-μl plates, non-treated, white, solid base
Corning 3852
1536
LEADseeker
Corning, 1536 10-μl non-treated, white, solid base
Corning 3937
1536
LEADseeker
Corning, TC-treated, sterile, white, solid base
Corning 3704
384
LEADseeker
Optiplate, white, solid base
Perkin Elmer 6005256
384
LEADseeker
Optiplate, white, solid base
Perkin Elmer 6005207
96
LEADseeker
Dynatech Microlite 1, white, solid base
Dynatech
96
LEADseeker
22
Ordering information Coating
Core bead type
Pack size
Streptavidin
PVT
50 mg 150 mg 500 mg 25 x 500mg 75 mg 250 mg 500 mg 50 mg 25 x 500 mg 500 mg 50 mg 25 x 500 mg 100 mg 500 mg 25 x 500 mg 250 mg 500 mg 50 mg 25 x 500 mg 500 mg 50 mg 25 x 500 mg 500 mg 500 mg 50 mg 500 mg 500 mg 50 mg 500 mg 500 mg 50 mg 1000 mg 500 mg 50 mg 500 mg 500 mg 500 mg 250 mg 125 mg 500 mg 50 mg 500 mg 50 mg 500 mg 50 mg 500 mg 500 mg 500 mg 50 mg 500mg 50mg 500mg 500mg 500 mg 500 mg
YSi PS YOx WGA
PVT YSi PS YOx
WGA PEI Type A WGA PEI Type B PEI (polyethyleneimine) Poly-L-lysine Membrane binding beads PDE Arginine binding beads Copper chelate Nickel chelate
PVT PS PVT PS PVT PS YSi PS YOx YSi YSi PVT YSi PS YOx
Glutathione
YSi
Protein A
PVT YSi YOx PS
Anti-mouse antibody
PVT YSi PS YOx
Product code SPA Scintillation SPA Imaging Beads Beads RPNQ0006 RPNQ0009 RPNQ0007 SPQ0032 RPNQ0015 RPNQ0012 RPNQ0261 RPNQ0263 RPNQ0285 RPNQ0271 RPNQ0273 RPNQ0283 RPNQ0252 RPNQ0001 SPQ0031 RPNQ00011 RPNQ0260 RPNQ0262 RPNQ0281 RPNQ0270 RPNQ0272 RPNQ0282 RPNQ0003 RPNQ0287 RPNQ0286 RPNQ0004 RPNQ0289 RPNQ0288 RPNQ0097 RPNQ0098 RPNQ0297 RPNQ0010 RPNQ0294 RPNQ0295 RPNQ0280 RPNQ0150 RPNQ0101 RPNQ0095 RPNQ0096 RPNQ0266 RPNQ0267 RPNQ0276 RPNQ0277 RPNQ0034 RPNQ0033 RPNQ0019 RPN143 RPNQ0274 RPNQ0275 RPNQ0264 RPNQ0265 RPNQ0017 RPN141 RPNQ0298 RPNQ0300 23
Coating
Core bead type
Pack size
Anti-rabbit antibody
PVT YSi PS YOx PVT YSi YOx PVT and YSi PS and YOx
500 mg 500 mg 500 mg 500 mg 500 mg 500 mg 500 mg
Anti-sheep antibody Charge-based binding SPA Scintillation Select-a-Bead Kit* SPA Imaging Select-a-Bead Kit†
Product code SPA Scintillation SPA Imaging Beads Beads RPNQ0016 RPN140 RPNQ0299 RPNQ0301 RPNQ0018 RPN142 RPNQ0320 RPNQ0250 RPNQ0291
* Contains 100 mg each of WGA PVT; WGA YSi; WGA PEI Type A, WGA PEI Type B, and Poly-L-lysine to allow quick and convenient receptor assay development using SPA Scintillation Beads. † Contains 50 mg each of WGA PS; WGA YOx; WGA PEI Type A, WGA PEI Type B, and Poly-L-lysine to allow quick and convenient receptor assay development using SPA Imaging Beads.
Related products These SPA screening kits are designed for use with SPA Scintillation Beads and do not contain enzyme unless specifically stated. Product
Quantity
Contents
Cholesterol Ester Transfer Protein (CETP) [3H] SPA (includes enzyme)
500 assay points
Cholesterol Ester Transfer Protein (CETP) [3H] SPA
500 assay points
[3H] PVT SPA Color Quench Kit
Sufficient for one uniformity check or one color quench correction curve.
• [3H]Cholesteryl ester HDL TRKQ7005-25UCI • Stop reagent (contains SPA Scintillation Beads) • Biotin LDL • Assay buffer • CETP (human) • Protocol • [3H]Cholesteryl ester HDL TRKQ7015-25UCI • Stop reagent (contains SPA Scintillation Beads) • Biotin LDL • Assay buffer • Protocol • [3H]PVT SPA Scintillation Beads TRKQ7080 • Tartrazine solution • Protocol
[125I] PVT SPA Color Quench Kit
Sufficient for one uniformity check or one color quench correction curve.
• [125I]PVT SPA Scintillation Beads • Tartrazine solution • Protocol
Farnesyl Transferase [3H] SPA Enzyme Assay
500 assay points
Helicase [3H] SPA Enzyme Assay
500 assay points
• Assay buffer TRKQ7010-100UCI • Bead reconstitution buffer • Biotin lamin B sequence peptide • Stop reagent • [3H]Farnesyl pyrophosphate (100 μCi) • Streptavidin SPA Scintillation Beads • Protocol • [3H]Helicase substrate solution (40 μCi) TRKQ7030-40UCI • Stop/capture solution • Streptavidin Scintillation SPA Beads in diluent buffer • Substrate dilution buffer • Assay buffer • Protocol
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Product code
RPAQ4030-56UCi
Product
Quantity
Contents
Product code
HIV-1 Integrase [3H] SPA Enzyme Assay
500 assay points
TRKQ7020-60UCI
Nitric Oxide Synthase Screening Kit
500 assay points
p34cdc2 [33P] Protein Kinase Enzyme Assay
500 assay points
Phosphodiesterase [3H]cAMP SPA Enzyme Assay
500 assay points
Phosphodiesterase [3H]cGMP SPA Enzyme Assay
500 assay points
Phospholipase A 2 [3H] SPA
500 assay points
[3H] Quan-T-RT
200 assay points
Serine/Threonine Protein Kinase SPA [33P] Enzyme Assay
500 assay points
Thymidine Uptake Assay
500 assay points
Biotinylated Myelin Basic Protein (bMBP)
25 μM
• [3H]DNA oligonucleotide • Denaturing solution • Assay buffer • Streptavidin SPA Scintillation Beads (lyophilized) • Manganese chloride solution • Bead reconstitution solution • Stop solution • Protocol • [3H]Arginine (70 μCi) • Arginine binding YSi SPA Scintillation Beads • Protocol • Assay buffer (includes biotin-tagged peptide) • Streptavidin PVT SPA Scintillation Beads • Stop solution • Protocol • [3H]cAMP • SPA Scintillation Beads • Assay buffer • Protocol • [3H]cGMP • SPA Scintillation Beads • Assay buffer • Protocol • Phospholipase A2 [3H] SPA substrate • Phospholipid mixed micelles • Assay buffer • Stop reagent/bead resuspension buffer • Streptavidin SPA Scintillation Beads • Protocol • Annealed primer/ template, bound to • Streptavidin SPA Scintillation Beads • Assay buffer • Stop solution • [3H]TTP (100 μCi) • Protocol • Assay buffer (including biotinylated substrate • Streptavidin PVT SPA Scintillation Beads • Stop solution • Protocol • SPA Scintillation Beads • Lysis buffer • Enhancer • Protocol • Supplied frozen, in buffer
100 μM
TRKQ7160-70UCI
RPNQ0170
TRKQ7090
TRKQ7100
TRKQ7040
TRK1022
RPNQ0200
RPNQ0130
RPNQ0201-2.5ml RPNQ0201-10ml RPNQ0202-1ml* RPNQ0202-10ml RPNQ0202-50ml
*bMBP: 1 ml is typically sufficient for ~ 200 assay points.
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www.gehealthcare.com GE Healthcare Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK
General Electric Company reserves the right, subject to any regulatory approval if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your GE Representative for the most current information. © 2005 General Electric Company - All rights reserved. GE and GE Monogram are trademarks of General Electric Company. LEADseeker, Cytostar-T, Amersham, and Amersham Biosciences are trademarks of GE Healthcare Limited. Cytostar-T is covered by US patent numbers 5665562 and5989854, European patent number 650396 B1, Japanese patent number 3152933, Finnish patent number 110488, and Canadian patent application number 2139409.Scintillation proximity assay technology is covered by European patent number 0154734 and by Japanese patent application number 84/52452. SPA technology using Imaging Beads is protected by Amersham Biosciences under US Patent Number 6,524,786, and foreign equivalents. MicroBeta and TopCount are trademarks of Perkin Elmer Life Sciences. Chameleon is a trademark of Hidex Oy. Amersham Biosciences UK Ltd, a General Electric company going to market as GE Healthcare.
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