Caution: For Laboratory Use. A product for research purposes only. Wheatgerm Agglutinin- PEI Type A, Type B PS SPA Imaging Beads

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1 SPA Beads Caution: For Laboratory Use. A product for research purposes only. Wheatgerm Agglutinin- PEI Type A, Type B PS SPA Imaging Beads Product Numbers: RPNQ0286 (Type A 50 mg) RPNQ0287 (Type A 500 mg) RPNQ0288 (Type B 50 mg) RPNQ0289 (Type B 500 mg) SAFETY WARNINGS AND PRECAUTIONS WARNING: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. CAUTION: For use with radioactive material. This product is to be used with radioactive material. Please follow the manufacturer s instructions relating to the handling, use, storage and disposal of such material. All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. HANDLING PACKAGING AND STORAGE Wheatgerm agglutinin SPA imaging beads are supplied as 50 mg or 500 mg lyophilized solid containing 10% sucrose by weight. This material should be stored, protected from light, at 2 8 C. EXPIRATION The expiration date is least >/= 4 weeks from the date of dispatch. QUALITY CONTROL Each batch of wheatgerm agglutinin (WGA) PEI Type A or B SPA Imaging beads is tested for its relative binding capacity of [ 3 H]N,N,N -triacetylchitotriose and [ 3 H methyl,1,2] thymidine triphosphate.

2 BEAD RECONSTITUTION Before use the beads should be reconstituted in a buffer appropriate for the particular assay to be performed. Standard buffers such as Tris, PBS and HEPES have all been routinely used in receptor SPA Imaging applications. The WGA-PEI Type A or B SPA Imaging beads are stable in a range of ph and solvents when used at typical assay concentrations i.e. <10% (v/v). However, it remains the responsibility of the user to evaluate the effect of the buffer composition and ph on the assay. The WGA- PEI Type A or B SPA Imaging beads should be mixed to ensure a homogeneous suspension whilst pipetting. This may be done by either recapping and shaking the bottle at regular intervals or by gently stirring with a magnetic stirrer. Reconstituted beads can usually be stored at 2 8 C for up to seven days. DO NOT FREEZE. PLEASE NOTE: The WGA-PEI Type A or B SPA Imaging beads have been freeze-dried from a 1% sucrose solution. Antimicrobial agents are not included in this reagent. The user should therefore be aware that microbial contamination might occur when the reconstituted beads are stored for prolonged periods. If antimicrobial agents (e.g. Sodium Azide) are added on storage, then it remains the responsibility of the user to evaluate the effects of the added agent on the assay. ASSAY PRINCIPLE WGA-PEI Type A or B SPA Imaging beads are designed to be used in receptor ligand binding assays. During assay incubation carbohydrate residues present in cell membranes bind to WGA on the SPA Imaging bead, effectively immobilizing the receptor-bearing membranes on to the SPA Imaging bead. The binding of radiolabelled ligands to such immobilized receptors brings the isotope into close proximity with the scintillant, which is incorporated within the bead. This allows the emitted radiation (beta-particles for [ 3 H] or Auger electrons for [ 125 I]) to stimulate the scintillant to emit light. Any unbound radiolabelled ligand is not in close enough proximity to the scintillant to allow such energy transfer and hence no signal is generated. Light emitted by stimulated SPA Imaging beads can be detected by imaging systems, with suitable filters. Schematic representation of receptor binding SPA imaging assay principle (1) Cold ligand or compound binds to the receptor captured onto the SPA Imaging bead. No signal is generated. (2)No radioligand binds to the receptor: no signal is generated. (3) Radioligand binds to the receptor

3 captured onto the SPA Imaging bead bringing the radioisotope into close proximity with the scintillant within the bead, stimulating the bead to emit light. Type A SPA Imaging beads are treated with wheatgerm agglutinin and then coated with polyethyleneimine (PEI). Type B SPA Imaging beads are treated with PEI and then coated with wheatgerm agglutinin. Determination of the amount of WGA-PEI Type A or B SPA Imaging bead required for a receptor binding assay To achieve optimal assay signal an excess of WGA-PEI Type A or B SPA Imaging bead should be present to ensure capture of all of the receptors present in the assay well. The amount of receptor preparation together with the radiolabelled ligand being used needs to be optimized for each assay. It remains the responsibility of the user to optimize the amount of WGA-PEI Type A or B SPA Imaging bead and the incubation time required for each assay. The following protocol outlines the steps required to optimize the amount of WGA-PEI Type A or B SPA Imaging bead and receptor required to generate an acceptable assay signal and background. An initial experiment will be required to determine the optimum amount of membrane and bead in the assay in 384 well plate format. The parameters to be measured are total signal (B 0 ), non-specific binding (NSB), and signal:background ratio. Generally B 0 signal increases as the amount of bead increases but NSB will also increase, potentially to the detriment of signal:background. (a) Use constant radioligand concentration and constant assay volume. (b) Include a no membrane control at each concentration of bead tested to observe NSB directly to bead. (c) Set up B 0 and NSB wells at each combination of membrane and bead to be tested. For iodinated ligands try mg of SPA Imaging bead per well and 1 5 µg of membrane protein. For tritiated ligands, try mg of SPA Imaging bead per well and µg of membrane protein. (d) For precoupled bead assays, the procedure of optimizing the membrane to bead involves precoupling increasing amounts of membrane to a fixed amount of bead, washing away any excess membrane and then testing a fixed aliquot (0.1 mg or 1 mg) in an assay with a fixed amount of radioligand to determine total and non-specific binding. (e) Image the fully equilibrated assay and calculate specific signal and NSB. A suggested imaging time is 5 minutes with coincident averaging, but it remains the user s responsibility to determine optimum imaging conditions. (f) Plotting the specific signal at each combination of bead and membrane should indicate when a bead is saturated with membrane. Examination of the NSB figures will show which bead: membrane ratio produces the best signal:background. TYPICAL DATA Melanocortin 4 receptor binding assay in 384 well microplate format utilizing WGA-PEI SPA Imaging beads MC 4 (melanocortin, subtype 4) receptors have been linked in several studies to the central regulation of appetite 1,2. Evidence for the role of MC 4 receptors in the central control of feeding comes from studies in MC 4 receptor knockout mice, which show an obese phenotype 2. Recent evidence suggests

4 melanocortins are involved in mediating the effects of leptin (appetite suppressant) and controlling the expression of neuropeptide Y (appetite stimulant), therefore the MC 4 receptor represents a potential target for anti-obesity drugs. A high-throughput screening assay has been developed to measure MC 4 receptor binding. SPA technology has been used to develop the assay in 96 and 384-well formats, and imaging technology has been used to develop the assay in 384 and 1536-well formats. ASSAY PROTOCOL B0 NSB Bkg Buffer 20 µl 10 µl 40 µl [Nle 4 D-Phe 7 ] α-melanocyte 10 µl stimulating hormone Membrane 10 µl 10 µl [ 125 I] [Tyr 2 ][Nle 4 D-Phe 7 ] α-melanocyte 10 µl 10 µl stimulating hormone PS Imaging bead 10 µl 10 µl 10 µl All reagents were pipetted into the plate, shaken for 30 minutes and incubated overnight at room temperature. The assays were imaged for 5 minutes, 3 3 binning with coincident averaging on the ViewLux Homogeneous Imaging System, ViewLux Multimodality Imaging System or ViewLux Radiometric Imaging System. Signal: background Total counts bound (B 0 ), non-specific binding (NSB) and signal:background ratio. Refer to assay protocol for assay conditions. PS WGA-PEI Type A SPA Imaging bead mean IOD s (n=3) signal:background Totals NSB 20.5 PS WGA-PEI Type B SPA Imaging bead mean IOD s (n=3) signal:background 214 Totals NSB 23. 5

5 PS WGA SPA Imaging beads 250 Type A 200 Type B 150 IOD,s Totals NSB Competition binding studies in 384 well microplates utilizing WGA-PEI REFERENCES 1. CHIESI, M. et al., Trends in Pharmacol. Sci., 22, (5), , (2001). 2. SCHWARTZ, M.W. et al., Nature, 404, , (2000). PerkinElmer, Inc. 549 Albany Street Boston, MA USA P: (800) or (+1) For a complete listing of our global offices, visit Copyright 2010, PerkinElmer, Inc. All rights reserved. PerkinElmer is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners.