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The DELFIA® Ligand Familyconsists of Europium-Labeled

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The DELFIA® Ligand Familyconsists of Europium-Labeled DELFIA Ligands Guide ® The DELFIA ligand family consists of Europium-labeled peptides and proteins designed to be used in ligand-receptor binding assays. DELFIA is based on time-resolved fluorescence lanthanide chemistry, known to be very sensitive and able to detect as low as 1 attomoles of Europium-labeled compound per well. This makes DELFIA a powerful, non-radioactive alternative for demanding ligand-receptor binding studies. DELFIA technology is based on the use of lanthanide chelate labels with unique fluorescent properties. The fluorescence lifetime of the special signal is several orders of magnitude longer than the non-specific background. This enables the label to be measured at a time when the background has already decayed. The large difference between excitation and emission wavelengths, and the narrow emission peak contribute to increasing the signal to background ratio. The sensitivity is furthermore increased because of the dissociation enhanced principle: the lanthanide chelate is dissociated into a new highly fluoresecent chelate inside a protective micelle. DELFIA Ligands for G-Protein Features Benefits Coupled Receptors Allows use of recombinant and High sensitivity endogeneous receptor membranes; DELFIA ligands are peptides and proteins labeled with DELFIA even with low expression levels europium-N1 chelate coupled to the amino end and/or lysine Non-radioactive No radioactive waste groups of the protein with the exception of motilin and NDP- αMSH to which the chelate is coupled to carboxyl end of the Long shelf life Allows flexibility in daily planning and budgeting peptide. The purity of the membrane Heterogeneous assay preparation is not as crucial as Cytokines Tachycines Growth Factors Others in homogeneous assays IL-2 Substance P EGF Motilin Membrane and whole Increased flexibility and throughput IL-4 Neurokinin A Galanin cell assays both in 96- and 384-well formats IL-5 Neurotensin IL-8 Bombesin Multiplexing capability Provides more information at lower cost TNFα NDP-αMSH The DELFIA ligand family is constantly growing, so please contact your local sales representative or visit us on the web at www.perkinelmer.com for any updates. For custom labeling, please email [email protected]. Stability DELFIA ligands are supplied in a lyophilized form and are stable Please note that each receptor may have a slightly different optimum for at least 1 year at 4°C. The exceptions to this are IL-2, IL-4, IL-5 buffer composition. Examples of optimized buffers for filtration and TNF-α, which are supplied in liquid form. Reconstituted assays are given in table 1. DELFIA ligands are stable for at least one month at -20°C and at DELFIA Assay Buffer (cat # 1244-106, 1244-111 and 4002-0010) can least 5 days at +4°C. be used for soluble receptor assays on coated plates, however, it is not recommended to be used in filtration based assays because it: DELFIA Ligand-Receptor Binding Assay Formats Contains inert red dye which may cause clogging in the filtration step DELFIA ligands can be used in several assay formats: Contains Tween 40 which harms cells and membranes TM As a filtration assay using AcroWell Filter Plate Doesn’t contain any divalent cation which is necessary As a solid phase assay using streptavidin coated microtitration for many ligand binding assays plates and biotinylated WGA As adherent cells on a cell culture plates (i.e Isoplates) Wash Buffer During the development phase, the biological activity of the Eu-labeled ligand has been demonstrated on AcroWell filtration The washing step should allow efficient removal of any free Eu-label based assays, with the exception of IL-2, IL-4, IL-5 and TNF-α. without disturbing the ligand-receptor complex. The streptavidin coated microplate format places more stringent DELFIA L*R wash concentrate (cat # CR135-250) contains TRIS-buffer demands on the receptor expression level. As a rule, the Bmax should supplemented with MgCl2 which is used in many ligand binding be >0.5 pmol/mg of protein when considering the streptavidin assays. It can be used in any DELFIA ligand-receptor binding assay. coated assay format. When using ≤ 0.5 pmol/mg, the filtration format is recommended. With AcroWell Filter Plates, up to 20 µg of protein DELFIA Wash Concentration (cat # 1244-114 and 4010-0010) can be can usually be used without clogging the plate filter. With adherent used in soluble receptor assays on coated plates. cells, we recommend to shake the plates to prevent the clogging of the filter. Optimizing the number of cells per well is recommended as well as using a gentle washing step to improve the variation of DELFIA Filtration Assay Protocol the assay. 25 µl Add unlabeled ligand (non-specific binding) Assay Buffer (5 µl) or buffer (total binding) to AcroWells DELFIA L*R binding buffer concentrate (cat # CR134-250) is 25 µl Add Eu-labeled ligand optimized to be used with AcroWell filtration assays: (5 µl) 50 mmol/L TRIS-HCl, pH 7.5 50 µl (10 µl) Add receptor 5 mmol/L MgCl2 25 µmol/L EDTA Incubate 90 min at RT 0.2 % BSA 300 µl Wash 4x under (100 µl) vacuum DELFIA L*R binding buffer concentrate: Prevents ligands from adsorbing to the well matrix, providing 100 µl Add Enhancement Incubate 15 min Read low assay background (20 µl) Solution shaking at RT or w/o on TRF shaking 45 min reader Prevents trace amounts of chelating agents from competing with the Europium ion, providing maximum thermal stability of the assay Figure 1. Schematic protocol for DELFIA ligand-receptor filtration Available at 10x concentrate to allow the addition of necessary assay displacement curve. Assay volumes shown in brackets are buffer additives to each receptor, e.g. protease inhibitors recommended to be used with AcroPrepTM 384 Filter Plate (Pall Life Sciences). This protocol has shown to be optimum for the Contains Mg2+ as divalent cation which is suitable for most DELFIA ligands and receptors shown in Table 1, except for receptors NDP-αMSH which should be incubated 120 minutes at 35 °C. Table 1. Examples of Kd and Ki values and signal-to-background (S/B) ratios received with the indicated receptor membrane preparation when using AcroWell 96-well filtration plates and buffers shown in the table. Similar Ki and Kd values have been obtained with AcroPrep 384 filtration plates (data not shown) B µg/well of Buffer addi- Receptor max K K tives added to DELFIA L*R Eu-ligand Receptor membrane fmol/well d i S/B source (pmol/mg) -1 DELFIA L*R wash buffer protein of receptor (nmol/L) (nmol/L) of protein binding buffer RBHMOTM expressed 1 % PVA Motilin recomb. hMot in HEK-293 cells 18.4 0.5 9.2 0.4 0.5 11 (Polyvinyl alcohol, yes (PerkinElmer) MW 6000) RBHCX2M expressed 2 mmol/L Interleukin-8 recomb. hCXCR2 in HEK 293 EBNA 1.7 3 5.1 1.9 0.5 8 CaCl2 yes cells (PerkinElmer) 50 µg/mL saponin RBHGL2M expressed 0.2 % Bacitracin Galanin recomb. hGalR2 in CHO cells 3-6 1 3-6 2.6 2.4 15 1 mol/L phenylmethyl- yes (PerkinElmer) sulfonyl fluoride RBHEGFM expressed 130 mmol/L NaCl endog. hEGFR in A431 cells EGF 11 1 11 0.8 0.4 12 50 µg/mL saponin yes (PerkinElmer) RBXNT1M expressed Neurotensin recomb. hNT1 in HEK-293 cells 1.06 5.7 6 1.1 0.3 7 none yes (PerkinElmer) 6110510 expressed 1 mmol/L Neurokinin A recomb. hNK2 in CHO cells 2.8 1 2.8 1.7 0.4 13 phenylmethylsulfonyl yes (Amersham) fluoride 6110551 expressed Substance P endog. hNK1 in UC11 cells 0.25 12 3 0.6 0.5 7 0.025% Bacitracin yes (Amersham) recomb hMC3 RBXMC3M 1.95 1 1.95 0.45 0.13 14 1 mmol/L yes NDP-αMSH recomb hMC4 RBHMC4M 1.63 2 3.26 0.6 0.27 15 phenylmethylsulfonyl yes recomb hMC5 RBXMC5M expressed 12.2 0.5 6.1 1.1 0.22 15 fluoride yes in HEK293 cells and 0.5 mmol/L (PerkinElmer) CaSO4 Bombesin recomb. hBB1 RBHBS1M expressed 3.4 0.4 1.36 0.5 0.24 4 yes in CHO cells (PerkinElmer) 50 µg/mL saponin recomb. hBB2 RBHBS2M expressed 9.3 0.2 1.86 0.5 0.2 8 yes in HEK293 cells (PerkinElmer) 1 Eu-ligand concentration is close to the Kd value in displacement studies. Table 2. DELFIA ligand-receptor assay comparison to radioactive filtration assay. K (nmol/L) K (nmol/L)-1 K (nmol/L)-1 K (nmol/L) ligand d i i d DELFIA assay DELFIA assay rad assay rad assay Multiplexing Capability Motilin 0.4 0.5 1.5 0.24 The unique chemical properties of Interleukin-8 1.9 0.5 0.19 0.29 lanthanides allow you to measure up to four ligand-receptor binding events Galanin 2.6 2.4 – 0.2 – 0.3 within the same well. High signal to background value is achieved by using EGF 0.8 0.4 5.8 0.8 both temporal and spectral resolution. When optimizing the multilabel assay, Neurotensin 1.1 0.3 0.25 0.23 please contact technical support for Neurokinin A 1.7 0.4 2.2 0.6 – 0.9 more detailed information (in Europe: [email protected], Substance P 0.6 0.5 0.02-0.5 0.2 in the U.S. and the rest of the world: [email protected]). To get NDP-αMSH MC3 0.45 0.13 0.44 0.22 MC4 0.6 – 1.1 0.27 1.0 0.15 Samarium (Sm) and Terbium (Tb) MC5 1.1 0.22 3.8 1.5 labeled ligands, please email [email protected].
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